NAC (NAM, ATAF1/2, and CUC2) transcription factors compose one of the largest families of plant-specific transcription factors; they are widely involved in plant growth and development and have especially important roles in improving stress resistance in plants. However, NAC gene family members in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed genome-wide due to the complexity of the alfalfa reference genome. In this study, a total of 421 M. sativa NAC genes (MsNACs) were identified from the alfalfa “Xinjiangdaye” reference genome. Basic bioinformatics analysis, including characterization of sequence length, protein molecular weight and genome position and conserved motif analysis, was conducted. Expression analysis showed that 47 MsNACs had tissue-specific expression, and 64 MsNACs were expressed in all tissues. The transcriptomic profiles of the genes were very different, indicating that these MsNACs have various functions in alfalfa growth and development. We identified 25, 42 and 47 MsNACs that respond to cold, drought and salt stress based on transcriptome data analysis and real-time quantitative PCR (RT–qPCR). Furthermore, 22 MsNACs were found to respond to both salt and drought stress, and 15 MsNACs were found to respond to cold, salt and drought stress. The results of this study could provide valuable information for further functional analysis of MsNACs and for the improvement of stress resistance in alfalfa.

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Heat shock transcription factors (HSFs) are important regulatory factors in plant stress responses to various biotic and abiotic stresses and play important roles in growth and development. The HSF gene family has been systematically identified and analyzed in many plants but it is not in the tetraploid alfalfa genome. We detected 104 HSF genes (MsHSFs) in the tetraploid alfalfa genome (“Xinjiangdaye” reference genome) and classified them into three subgroups: 68 in HSFA, 35 in HSFB and 1 in HSFC subgroups. Basic bioinformatics analysis, including genome location, protein sequence length, protein molecular weight and conserved motif identification, was conducted. Gene expression analysis revealed tissue-specific expression for 13 MsHSFs and tissue-wide expression for 28 MsHSFs. Based on transcriptomic data analysis, 21, 11 and 27 MsHSFs responded to drought stress, cold stress and salt stress, respectively, with seven responding to all three. According to RT–PCR, MsHSF27/33 expression gradually increased with cold, salt and drought stress condition duration; MsHSF6 expression increased over time under salt and drought stress conditions but decreased under cold stress. Our results provide key information for further functional analysis of MsHSFs and for genetic improvement of stress resistance in alfalfa.

Salt stress is a worldwide agronomic issue that limits crop yield and quality. Improving salt stress tolerance via genetic modification is the most efficient method to conquer soil salinization problems in crops. Crop miRNAs have been declared to be tightly associated with responding and adapting to salt stress and are advantageous for salt tolerance modification. However, very few studies have validated vital salt tolerance miRNAs and coupled potent target genes in Medicago species, the most economically important forage legume species. In this study, Mtr-miR319a, a miRNA that was identified from the previous next-generation sequencing assay of salt-treated Medicago truncatula, was overexpressed in M. truncatula and Arabidopsis thaliana, inducing the curly leaves and salt stress tolerance phenotypes. Combining the elevated expression level of Mtr-miR319a in the M. truncatula overexpression lines under normal and salt-treatment conditions, the regulatory roles of Mtr-miR319a in leaf development and salt stress adaptation were demonstrated. Several predicted target genes of Mtr-miR319a were also regulated by Mtr-miR319a and were associated with the aforementioned phenotypes in M. truncatula plants, most notably MtTCP4. Our study clarified the functional role of Mtr-miR319a and its target genes in regulating leaf development and defending salt stress, which can help to inform crop breeding efforts for improving salt tolerance via genetic engineering.