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Epigenetic modifications play important roles in genome evolution and innovation. However, most analyses have focused on the evolutionary role of DNA modifications, and little is understood about the influence of posttranscriptional RNA modifications on genome evolution. To explore the evolutionary significance of RNA modifications, we generated transcriptome-wide profiles of N6-methyladenosine (m6A), the most prevalent internal modification of mRNA, for 13 representative plant species spanning over half a billion years of evolution. These data reveal the evolutionary conservation and divergence of m6A methylomes in plants, uncover the preference of m6A modifications on ancient orthologous genes, and demonstrate less m6A divergence between orthologous gene pairs with earlier evolutionary origins. Further investigation revealed that the evolutionary divergence of m6A modifications is related to sequence variation between homologs from whole-genome duplication and gene family expansion from local-genome duplication. Unexpectedly, a significant negative correlation was found between the retention ratio of m6A modifications and the number of family members. Moreover, the divergence of m6A modifications is accompanied by variation in the expression level and translation efficiency of duplicated genes from whole- and local-genome duplication. Our work reveals new insights into evolutionary patterns of m6A methylomes in plant species and their implications, and provides a resource of plant m6A profiles for further studies of m6A regulation and function in an evolutionary context.

RNA N6-methyladenosine (m6A) modification is the most abundant form of RNA epigenetic modification in eukaryotes. Given that m6A evolution is associated with the selective constraints of nucleotide sequences in mammalian genomes, we hypothesize that m6A evolution can be linked, at least in part, to genomic duplication events in complex polyploid plant genomes. To test this hypothesis, we presented the maize (Zea mays) m6A modification landscape in a transcriptome-wide manner and identified 11,968 m6A peaks carried by 5,893 and 3,811 genes from two subgenomes (maize1 and maize2, respectively). Each of these subgenomes covered over 2,200 duplicate genes. Within these duplicate genes, those carrying m6A peaks exhibited significant differences in retention rate. This biased subgenome fractionation of m6A-methylated genes is associated with multiple sequence features and is influenced by asymmetric evolutionary rates. We also characterized the coevolutionary patterns of m6A-methylated genes and transposable elements, which can be mediated by whole genome duplication and tandem duplication. We revealed the evolutionary conservation and divergence of duplicated m6A functional factors and the potential role of m6A modification in maize responses to drought stress. This study highlights complex interplays between m6A modification and gene duplication, providing a reference for understanding the mechanisms underlying m6A evolution mediated by genome duplication events.

RNA N(6)-methyladenosine (m(6)A) modification is the most abundant form of RNA epigenetic modification in eukaryotes. Given that m(6)A evolution is associated with the selective constraints of nucleotide sequences in mammalian genomes, we hypothesize that m(6)A evolution can be linked, at least in part, to genomic duplication events in complex polyploid plant genomes. To test this hypothesis, we presented the maize (Zea mays) m(6)A modification landscape in a transcriptome-wide manner and identified 11,968 m(6)A peaks carried by 5,893 and 3,811 genes from two subgenomes (maize1 and maize2, respectively). Each of these subgenomes covered over 2,200 duplicate genes. Within these duplicate genes, those carrying m(6)A peaks exhibited significant differences in retention rate. This biased subgenome fractionation of m(6)A-methylated genes is associated with multiple sequence features and is influenced by asymmetric evolutionary rates. We also characterized the coevolutionary patterns of m(6)A-methylated genes and transposable elements, which can be mediated by whole genome duplication and tandem duplication. We revealed the evolutionary conservation and divergence of duplicated m(6)A functional factors and the potential role of m(6)A modification in maize responses to drought stress. This study highlights complex interplays between m(6)A modification and gene duplication, providing a reference for understanding the mechanisms underlying m(6)A evolution mediated by genome duplication events.

Cold acclimation is an important process by which plants respond to low temperature and enhance their winter hardiness. C-REPEAT BINDING FACTOR1 (CBF1), CBF2, and CBF3 genes were shown previously to participate in cold acclimation in Medicago truncatula In addition, MtCBF4 is transcriptionally induced by salt, drought, and cold stresses. We show here that MtCBF4, shown previously to enhance drought and salt tolerance, also positively regulates cold acclimation and freezing tolerance. To identify molecular factors acting upstream and downstream of the MtCBF4 transcription factor (TF) in cold responses, we first identified genes that are differentially regulated upon MtCBF4 overexpression using RNAseq Digital Gene Expression Profiling. Among these, we showed that MtCBF4 directly activates the transcription of the COLD ACCLIMATION SPECIFIC15 (MtCAS15) gene. To gain insights into how MtCBF4 is transcriptionally regulated in response to cold, an R2R3-MYB TF, MtMYB3, was identified based on a yeast one-hybrid screen as binding directly to MYB cis-elements in the MtCBF4 promoter, leading to the inhibition of MtCBF4 expression. In addition, another MYB TF, MtMYB61, identified as an interactor of MtMYB3, can relieve the inhibitory effect of MtMYB3 on MtCBF4 transcription. This study, therefore, supports a model describing how MtCBF4 is regulated by antagonistic MtMYB3/MtMYB61 TFs, leading to the up-regulation of downstream targets such as MtCAS15 acting in cold acclimation in M. truncatula.

• Dehydrins are a type of late embryogenesis abundant protein. Some dehydrins are involved in the response to various abiotic stresses. Accumulation of dehydrins enhances the drought, cold and salt tolerances of transgenic plants, although the underlying mechanism is unclear. MtCAS31 (Medicago Truncatula cold-acclimation specific protein 31) is a Y(2)K(4)-type dehydrin that was isolated from Medicago truncatula. • We analyzed the subcellular and histochemical localization of MtCAS31, and the expression patterns of MtCAS31 under different stresses. Transgenic Arabidopsis that overexpressed MtCAS31 was used to determine the function of MtCAS31. A yeast two-hybrid assay was used to screen potential proteins that could interact with MtCAS31. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay. • After a 3-h drought treatment, the expression of MtCAS31 significantly increased 600-fold. MtCAS31 overexpression dramatically reduced stomatal density and markedly enhanced the drought tolerance of transgenic Arabidopsis. MtCAS31 could interact with AtICE1 (inducer of CBF expression 1) and the AtICE1 homologous protein Mt7g083900.1, which was identified from Medicago truncatula both in vitro and in vivo. • Our findings demonstrate that a dehydrin induces decreased stomatal density. Most importantly, the interaction of MtCAS31 with AtICE1 plays a role in stomatal development. We hypothesize that the interaction of MtCAS31 and AtICE1 caused the decrease in stomatal density to enhance the drought resistance of transgenic Arabidopsis.

Background The entire world is facing a deteriorating environment. Understanding the mechanisms underlying plant responses to external abiotic stresses is important for breeding stress-tolerant crops and herbages. Phytohormones play critical regulatory roles in plants in the response to external and internal cues to regulate growth and development. Medicago falcata is one of the stress-tolerant candidate leguminous species and is able to fix atmospheric nitrogen. This ability allows leguminous plants to grow in nitrogen deficient soils. Methods We performed Illumina sequencing of cDNA prepared from abiotic stress treated M. falcata. Sequencedreads were assembled to provide a transcriptome resource. Transcripts were annotated using BLASTsearches against the NCBI non-redundant database and gene ontology definitions were assigned. Acomparison among the three abiotic stress treated samples was carried out. The expression of transcriptswas confirmed with qRT-PCR. Results We present an abiotic stress-responsive M. falcata transcriptome using next-generation sequencing data from samples grown under standard, dehydration, high salinity, and cold conditions. We combined reads from all samples and de novo assembled 98,515 transcripts to build the M. falcata gene index. A comprehensive analysis of the transcriptome revealed abiotic stress-responsive mechanisms underlying the metabolism and core signalling components of major phytohormones. We identified nod factor signalling pathways during early symbiotic nodulation that are modified by abiotic stresses. Additionally, a global comparison of homology between the M. falcata and M. truncatula transcriptomes, along with five other leguminous species, revealed a high level of global sequence conservation within the family. Conclusions M. falcata is shown to be a model candidate for studying abiotic stress-responsive mechanisms in legumes. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to abiotic stresses. Our data provides many gene candidates that might be used for herbage and crop breeding. Additionally, FalcataBase (http://bioinformatics.cau.edu.cn/falcata/) was built for storing these data. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2019-x) contains supplementary material, which is available to authorized users.

Background Medicago truncatula has been chosen as a model species for genomic studies. It is closely related to an important legume, alfalfa. Transporters are a large group of membrane-spanning proteins. They deliver essential nutrients, eject waste products, and assist the cell in sensing environmental conditions by forming a complex system of pumps and channels. Although studies have effectively characterized individual M. truncatula transporters in several databases, until now there has been no available systematic database that includes all transporters in M. truncatula. Description The M. truncatula transporter database (MTDB) contains comprehensive information on the transporters in M. truncatula. Based on the TransportTP method, we have presented a novel prediction pipeline. A total of 3,665 putative transporters have been annotated based on International Medicago Genome Annotated Group (IMGAG) V3.5 V3 and the M. truncatula Gene Index (MTGI) V10.0 releases and assigned to 162 families according to the transporter classification system. These families were further classified into seven types according to their transport mode and energy coupling mechanism. Extensive annotations referring to each protein were generated, including basic protein function, expressed sequence tag (EST) mapping, genome locus, three-dimensional template prediction, transmembrane segment, and domain annotation. A chromosome distribution map and text-based Basic Local Alignment Search Tools were also created. In addition, we have provided a way to explore the expression of putative M. truncatula transporter genes under stress treatments. Conclusions In summary, the MTDB enables the exploration and comparative analysis of putative transporters in M. truncatula. A user-friendly web interface and regular updates make MTDB valuable to researchers in related fields. The MTDB is freely available now to all users at http://bioinformatics.cau.edu.cn/MtTransporter/.