m6A RNA Methylation in Marine Plants: First Insights and Relevance for Biological Rhythms

Abstract

Circadian regulations are essential for enabling organisms to synchronize physiology with environmental light-dark cycles. Post-transcriptional RNA modifications still represent an understudied level of gene expression regulation in plants, although they could play crucial roles in environmental adaptation. N6-methyl-adenosine (m6A) is the most prevalent mRNA modification, established by “writer” and “eraser” proteins. It influences the clockwork in several taxa, but only few studies have been conducted in plants and none in marine plants. Here, we provided a first inventory of m6A-related genes in seagrasses and investigated daily changes in the global RNA methylation and transcript levels of writers and erasers in Cymodocea nodosa and Zostera marina. Both species showed methylation peaks during the dark period under the same photoperiod, despite exhibiting asynchronous changes in the m6A profile and related gene expression during a 24-h cycle. At contrasting latitudes, Z. marina populations displayed overlapping daily patterns of the m6A level and related gene expression. The observed rhythms are characteristic for each species and similar in populations of the same species with different photoperiods, suggesting the existence of an endogenous circadian control. Globally, our results indicate that m6A RNA methylation could widely contribute to circadian regulation in seagrasses, potentially affecting the photo-biological behaviour of these plants.

Daily Regulation of Key Metabolic Pathways in Two Seagrasses Under Natural Light Conditions

“…Such genes were also those making a greater contribution to sample distribution in the PCA (Figure 1 and Supplementary Table 4). Despite this common behavior, the variations we observed in their daily transcriptional timing, were species-specific (Table 2 and Supplementary Tables 5, 6), highlighting the importance of intrinsic biological, and likely ecological, attributes in determining the periodicity of functions that go beyond the (common) effects of external stimuli (Müller et al, 2014;Ruocco et al, 2020a). Five GOIs (i.e., SPS1, GWD, FD, LHY and LHCA1) showed a significant Ti×Sp interaction, indicating their daily pattern of expression was substantially different between the two seagrass species (Supplementary Table 6).…”

“…Seagrass specimens used for this study are the same as in Ruocco et al (2020a). In May 2019, entire shoots of Cymodocea nodosa and Zostera marina were collected by snorkeling from a shallow-water (1-2 m depth) mixed meadow near Ilha da Culatra (Faro, Portugal -36 • 59 41 N, 7 • 50 26 W) with a significant portion of the sediment.…”

“…The thermal profile of the reactions was as follows: 95 • C for 20 s, 40 times 95 • C for 1 s, and 60 • C for 20 s. All RT-qPCR reactions were conducted in triplicate, and each assay included three no-template negative controls. To normalize target gene expression, we used the eukaryotic initiation factor 4A (eIF4A) as a reference gene (RG), which was previously demonstrated to exhibit a stable expression in Z. marina and C. nodosa along a daily cycle (Rasmusson et al, 2017;Ruocco et al, 2020a) and under a range of different conditions (Ransbotyn and Reusch, 2006;Winters et al, 2011;Olivé et al, 2017). RT-qPCR efficiencies for all primer pairs were calculated from the slopes of the standard curves of the threshold cycle (CT) vs. cDNA concentration with the equation E = 10 −1/slope .…”

Current Insights into m6A RNA Methylation and Its Emerging Role in Plant Circadian Clock

“…In addition, cry1cry2 knockdown mice with a complete loss of circadian rhythm have significantly lower levels of m 6 A and a loss of the circadian rhythm of m 6 A [ 118 ]. In plants, daily oscillation of mRNA transcripts of the writers and erasers and accumulation of global m 6 A levels at midnight have been observed in seagrasses Cymodocea nodosa and Zostera marina [ 119 ].…”

Light, chromatin, action: nuclear events regulating light signaling in Arabidopsis

“…Interestingly, the function of RNA methylation in circadian clock entrainment by light appears to be evolutionarily conserved from plants to metazoans (Fustin et al ., 2013 ). The N6‐methyladenosine level peaks during the night in the seagrasses Cymodocea nodosa and Zostera marina (Ruocco et al ., 2020 ), and likewise m 6 A levels increase at night in mice liver cells (Wang et al ., 2015 ), suggesting that circadian oscillation of the epitranscriptome could be a conserved feature across kingdoms, yet this remains to be assessed in Arabidopsis. Similar to plants, deficiency in CRY‐dependent blue light perception in mammals decreases m 6 A transcript levels (Wang et al ., 2015 ) and human CRY2 can interact with the m 6 A writer complex subunits METTL3, METTL14 and WTAP (X. Wang et al ., 2021 ), suggesting the existence of conserved mechanisms connecting light signaling to epitranscriptomic regulations.…”