3.3. Transition to aquatic environment results in longer poly(A) tails and different non-adenine modification patterns to mRNA transcripts
The poly(A) tail is not a static, simple entity that merely denotes the 3’ end. Rather, the poly(A) tail should be viewed as a dynamic and variable part of the transcript . Polyadenylation, characterized by the addition of poly(A) tails to mRNA molecules, is a critical post-transcriptional modification influencing mRNA stability, nuclear export, and translation efficiency . In plants, the regulation of poly(A) tail length plays a pivotal role in responding to various stress conditions, thereby facilitating adaptive responses that ensure survival in changing environments . Poly(A) tails, by influencing the mRNA’s metabolic fate, act as a dynamic regulatory mechanism that can be modulated in response to stress, thus impacting gene expression patterns crucial for stress adaptation. Research has demonstrated that alternative polyadenylation (APA) leading to the generation of mRNA isoforms with differing poly(A) tail lengths, is a novel strategy for the regulation of gene expression in response to stresses in plants . APA contributes to the diversification of the transcriptome and proteome under stress conditions, enabling plants to fine-tune the expression of genes involved in stress responses (). For instance, inArabidopsis thaliana , the poly(A) tail length of specific mRNAs has been shown to vary in response to heat shock, suggesting that the modulation of poly(A) tail length is a mechanism through which plants respond to thermal stress by controlling the stability and translation of heat shock protein (HSP) mRNAs . This modulation ensures the rapid accumulation of HSPs, crucial for protein folding and protection under heat stress. Furthermore, the study of full-length RNA molecules across different tissues has revealed tissue-specific and evolutionarily conserved regulation of poly(A) tail length, indicating that this mechanism is fundamental to plant development and stress responses . Deep transcriptomic direct RNA analysis revealed information on 156 906 polyA tails in Riccia fluitans, with 50 694 being identified in terrestrial and 106 212 in aquatic form of plants (Supporting Information S1: Table 14). Globally, the elongation bias of poly(A) tails was observed in the aquatic form of Riccia fluitans (Figure 4C). Nine transcripts exhibited significant differences in tail length, including CL.26773.1 (transcript coding - galactose oxidase/kelch repeat superfamily protein, CL.12661.2 (hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase), CL.34006.3 (Enoyl-CoA hydratase/isomerase family), CL.20497.1 (UDP-glucosyl transferase 73B1) and two unknown (CL.33217.1 and CL.7501.1) (Supporting Information S1: Table 15). The mentioned transcripts displayed elongated tails in their terrestrial environment, while CL.22730.2 (coding ABC-2 type transporter family protein), CL.20863.3 (serine carboxypeptidase-like 20), and CL.34882.1 (Rab5-interacting family protein) in the aquatic condition (Figure 4A and 4B). The CL.22730.2 had the most tails isoform detected among statistically significant transcripts. In detail, 31 polyA tails were specific to aquatic form and 8 to the terrestrial variants (Figure 4E). Changes in polyA tail length can significantly impact also the ability to withstand water stress in Arabidopsis thaliana . The mRNAs with longer poly(A) tails are generally more stable and efficiently translated, leading to an increased accumulation of proteins essential for stress response . This adaptive strategy enhances the plant’s resilience to water stress by improving its water retention and stress signaling pathways, ultimately contributing to its survival under adverse environmental conditions. Further studies on the role of polyA tail length in environmental adaptations of early land plants could shed new light in the molecular processes behind terrestrialization. The influence of U and G non-A at the end of poly(A) tails on mRNA stability regulation has been demonstrated, where they can either inhibit or promote poly(A) tail degradation . In Arabidopsis thaliana , non-adenine nucleotides have been found in the polyA tail, suggesting that more uniform poly(a) tails in poly(A)-binding proteins may increase translation efficiency . We have shown that non-A modifications also occur in Riccia fluitans. 8884 non-a observations were detected in water and 4609 in land form. The most frequent non-A was cytosine with 3979 observations in water-form Riccia and guanine with 1976 observations in the land form (Figure 5A). The unknown CL.7154.1 was marked as the most abundant non-A transcript in the aquatic environment, while the unknown CL.7156.1 in the land environment. Summarized number of non-A events in both environmentals, the highest amount of non-A modifications were annotated in Cold, circadian rhythm, and rna binding 2 transcript (CL.11266.1) (Supporting Information S1: Table 16). Transcripts with non-a were involved in GO processes such as cytoplasm (GO:0005737), cytosol (GO:0005829), plastid (GO:0009536), organelle envelope (GO:0031967), and chloroplast (GO:0009507) (Figure 5B and 5C and Supporting Information S1: Table 17 and 18). It will be interesting to investigate the dynamics of poly(A) tails in this liverwort under environmental changes, as we see clear differences in tail lengths under environmental changes and a global change in the number and proportion of non-A mutations in poly(A) tails.