mRNA N⁶‐methyladenosine is critical for cold tolerance in Arabidopsis

Abstract

Plants respond to low temperatures by altering the mRNA abundance of thousands of genes contributing to numerous physiological and metabolic processes that allow them to adapt. At the post-transcriptional level, these cold stress-responsive transcripts undergo alternative splicing, microRNA-mediated regulation and alternative polyadenylation, amongst others. Recently, m 6 A, m 5 C and other mRNA modifications that can affect the regulation and stability of RNA were discovered, thus revealing another layer of posttranscriptional regulation that plays an important role in modulating gene expression. The importance of m 6 A in plant growth and development has been appreciated, although its significance under stress conditions is still underexplored. To assess the role of m 6 A modifications during cold stress responses, methylated RNA immunoprecipitation sequencing was performed in Arabidopsis seedlings esposed to low temperature stress (4°C) for 24 h. This transcriptome-wide m 6 A analysis revealed large-scale shifts in this modification in response to low temperature stress. Because m 6 A is known to affect transcript stability/ degradation and translation, we investigated these possibilities. Interestingly, we found that cold-enriched m 6 A-containing transcripts demonstrated the largest increases in transcript abundance coupled with increased ribosome occupancy under cold stress. The significance of the m 6 A epitranscriptome on plant cold tolerance was further assessed using the mta mutant in which the major m 6 A methyltransferase gene was mutated. Compared to the wild-type, along with the differences in CBFs and COR gene expression levels, the mta mutant exhibited hypersensitivity to cold treatment as determined by primary root growth, biomass, and reactive oxygen species accumulation. Furthermore, and most importantly, both non-acclimated and cold-acclimated mta mutant demonstrated hypersensitivity to freezing tolerance. Taken together, these findings suggest a critical role for the epitranscriptome in cold tolerance of Arabidopsis.

Recent advances in the plant epitranscriptome

“…R = A/G; W = A/U; K = G/U; Y = C/U; D = A/G/U; H = A/C/U; V = A/G/C. b m 6 A modifications are influenced by various endogenous and environmental signals as shown in the following studies: developmental stages (strawberry [ 117 ], wheat [ 125 ]); different organs ( Arabidopsis [ 137 ]); ecotype divergence ( Arabidopsis [ 138 ]); blue light ( Arabidopsis [ 79 ]); salt stress ( Arabidopsis [ 139 , 140 ], sweet sorghum [ 128 ], rice [ 141 ], sugar beet [ 142 ], cotton [ 133 ]); heat/low temperature (pak-choi [ 122 ], Arabidopsis [ 143 ], tomato [ 124 ]); cadmium stress (rice [ 127 ], barley [ 132 ]); drought stress (sea-buckthorn [ 118 ], populus [ 119 ], apple [ 135 , 144 ]); pathogen infection (apple [ 108 ], rice [ 145 ], wheat [ 126 ], watermelon [ 131 ], pear [ 116 ]); and nematode infection (soybean [ 146 ]). c Expression of m 6 A writer and eraser genes are modulated by multiple external stimuli in Arabidopsis .…”

N6‐Methyladenosine modification of mRNA contributes to the transition from 2D to 3D growth in the moss Physcomitrium patens

“…We first arranged these 79 recovered transcripts according to their differential expression, from up‐ to downregulated (33 and 46 mRNAs, respectively). When we observed the extent of methylation in each mRNA (as defined by the P ‐value), there was no clear association between this value and the direction of changes in mRNA accumulation (Figure 5a), suggesting that additional factors determine the fate of the m 6 A‐containing transcripts to direct them to down‐ or upregulation, as described in other biological systems (Frye et al., 2018; Ivanova et al., 2017; Wang et al., 2021), including plants (Anderson et al., 2018; Govindan et al., 2022). Among these 79 m 6 A‐DE transcripts we identified factors involved in the development of buds, for instance PpAPB2 (Pp3c9_25570) (Aoyama et al., 2012), and others related to auxin signaling, such as the ARF domain protein (Pp3c6_21370V3.1) and the AUX/IAA domain factor (Pp3c15_9710V3.1), which are methylated and downregulated in Ppmta plants (indicated by aqua asterisks in Figure 5a,c; Figure S7).…”

“…By applying meme algorithms to identify potential methylation sequence motifs enriched in these regions, we found a 7‐nt sequence with the consensus GRAGRAG (where R = A/G, P = 9.1e‐056; Figure 4c). Previous reports have shown that in plants the methylation motif RRACH has undergone diversification, and furthermore, depending on the tissue, growth conditions or environmental stimuli, the modified mRNAs exhibit different consensus motifs (Anderson et al., 2018; Govindan et al., 2022; Zhou et al., 2022). Here, we propose GRAGRAG as a methylation motif present in P .…”

“…The molecular mechanisms that underly the development of 3D growth are poorly understood, mainly because 3D growth in seed plants starts during embryo development, and therefore mutants exhibiting defects in 3D growth are lethal (Meinke & Sussex, 1979;Sun et al, 1998). The life cycle of the moss Physcomitrium patens recapitulates the transition from filamentous growth, to planar branching, to full 3D growth, and a similar transition occurs later during the development of its zygote (Cove & Knight, 1993;Govindan et al, 2022;Harrison et al, 2009). Nonetheless, mutations in genes involved in the transition from 2D to 3D growth are not lethal, as plants can survive by filamentous growth indefinitely (Moody et al, 2018), turning P. patens into an ideal model to study 3D growth modulation in plants.…”

“…In plants, the MTC has been identified and orthologs of METTL3, METTL14 and WTAP, named MTA, MTB and FIP37, respectively, have been studied, mostly in Arabidopsis thaliana (Bodi et al., 2012; Růžička et al., 2017; Shen et al., 2016; Zhong et al., 2008). The biological effects of m 6 A modification of mRNA in plants are diverse, including the regulation of organ growth and determination (Arribas‐Hernández et al., 2018; Růžička et al., 2017; Shen et al., 2016), embryo development (Zhong et al., 2008) and responses to environmental signals (Anderson et al., 2018; Govindan et al., 2022; Hu et al., 2021).…”