The AP2/ERF superfamily is a large, plant-specific transcription factor family that is involved in many important processes, including plant growth, development and stress responses. Using Medicago truncatula genome information, we identified and characterized 123 putative AP2/ERF genes, which were named as MtERF1–123. These genes were classified into four families based on phylogenetic analysis, which is consistent with the results of other plant species. MtERF genes are distributed throughout all chromosomes but are clustered on various chromosomes due to genomic tandem and segmental duplication. Using transcriptome, high-throughput sequencing data and qRT-PCR analysis, we assessed the expression patterns of the MtERF genes in tissues during development and under abiotic stresses. In total, 87 MtERF genes were expressed in plant tissues, most of which were expressed in specific tissues during development or under specific abiotic stress treatments. These results support the notion that MtERF genes are involved in developmental regulation and environmental responses in M. truncatula. Furthermore, a cluster of DREB subfamily members on chromosome 6 was induced by both cold and freezing stress, representing a positive gene regulatory response under low temperature stress, which suggests that these genes might contribute to freezing tolerance to M. truncatula. In summary, our genome-wide characterization, evolutionary analysis and expression pattern analysis of MtERF genes in M. truncatula provides valuable information for characterizing the molecular functions of these genes and utilizing them to improve stress tolerance in plants.

Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress affecting crop productivity. Alfalfa, a perennial forage legume, expresses some tolerance to biotic and abiotic stress, especially to saline-alkaline stress. To elucidate the mechanism underlying plant saline-alkaline tolerance, we conducted ion torrent sequencing and transcriptome analysis of whole alfalfa seedlings treated with saline-alkaline solutions. Transcriptome database was generated from 53,853 unigenes. Of 4,137 differentially expressed genes, 2,752 and 2,633 genes altered significantly their expression levels after 1 d and 7 d of treatment, respectively. Gene ontology analysis recognized 14 pathways highly enriched in biological process, metabolic, and signal pathway categories and demonstrated the differential response of metabolic pathways after both treatments. The expression levels of 109 and 96 transcription factors were altered significantly after 1 d and 7 d of treatment, respectively. Specific responses of peroxidase, flavonoid, and light pathway component indicate that the antioxidant capacity is one of the central mechanisms of saline-alkaline stress tolerance in alfalfa. Responses of eight genes specific to saline-alkaline stress were screened among 18 different expression genes comparing to salt stress-induced genes to uncover different mechanisms involved in plant tolerance to saline and alkaline stress. Among them, the expression levels of an NAC transcription factor and a glutathione S-transferase were altered significantly in treatments; they were upregulated under saline-alkaline stress and downregulated under salt stress. The transcriptome analysis in this study provides a novel insight into the understanding of saline-alkaline stress tolerance mechanisms in alfalfa.

Alfalfa (Medicago sativa L.) is an important perennial forage, with high nutritional value, which is widely grown in the world. Because of low freezing tolerance, its distribution and production are threatened and limited by winter weather. To understand the complex regulation mechanisms of freezing tolerance in alfalfa, we performed transcriptome sequencing analysis under cold (4 °C) and freezing (-8 °C) stresses. More than 66 million reads were generated, and we identified 5767 transcripts differentially expressed in response to cold and/or freezing stresses. These results showed that these genes were mainly classified as response to stress, transcription regulation, hormone signaling pathway, antioxidant, nodule morphogenesis, etc., implying their important roles in response to cold and freezing stresses. Furthermore, nine CBF transcripts differentially expressed were homologous to CBF genes of Mt-FTQTL6 site, conferring freezing tolerance in M. truncatula, which indicated that a genetic mechanism controlling freezing tolerance was conservative between M. truncatula and M. sativa. In summary, this transcriptome dataset highlighted the gene regulation response to cold and/or freezing stresses in alfalfa, which provides a valuable resource for future identification and functional analysis of candidate genes in determining freezing tolerance.

Winter damage, especially in northern climates, is a major limitation of the utilization of perennial forages such as alfalfa. Therefore, improving freezing tolerance is imperative in alfalfa genetic breeding. However, freezing tolerance is a complex trait that is determined by many genes. To understand the complex regulation mechanisms of freezing tolerance in alfalfa, we performed small RNA sequencing analysis under cold (4°) and freezing (−8°) stress. The sequencing results revealed that 173 known, and 24 novel miRNAs were expressed, and that the expression of 35 miRNAs was affected by cold and/or freezing stress. Meanwhile, 105 target genes cleaved by these miRNAs were characterized by degradome sequencing. These targets were associated with biological regulation, cellular processes, metabolic processes, and response to stress. Interestingly, most of them were characterized as transcription factors (TFs), including auxin response factors, SBP, NAC, AP2/ERF, and GRF, which play important roles in plant abiotic responses. In addition, important miRNAs and mRNAs involved in nodulation were also identified, for example, the relationship between miR169 and the TF CCAAT (also named as NF-YA/HAP2), which suggested that nodulation has an important function in freezing tolerance in alfalfa. Our results provide valuable information to help determine the molecular mechanisms of freezing tolerance in alfalfa, which will aid the application of these miRNAs and their targets in the improvement of freezing tolerance in alfalfa and related plants.