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Background: Jasmonate ZIM-domain (JAZ) proteins, which act as negative regulators in the jasmonic acid (JA) signalling pathway, have significant implications for plant development and response to abiotic stress. Results: Through a comprehensive genome-wide analysis, a total of 20 members of the JAZ gene family specific to alfalfa were identified in its genome. Phylogenetic analysis divided these 20 MsJAZ genes into five subgroups. Gene structure analysis, protein motif analysis, and 3D protein structure analysis revealed that alfalfa JAZ genes in the same evolutionary branch share similar exon‒intron, motif, and 3D structure compositions. Eight segmental duplication events were identified among these 20 MsJAZ genes through collinearity analysis. Among the 32 chromosomes of the autotetraploid cultivated alfalfa, there were 20 MsJAZ genes distributed on 17 chromosomes. Extensive stress-related cis-acting elements were detected in the upstream sequences of MsJAZ genes, suggesting that their response to stress has an underlying function. Furthermore, the expression levels of MsJAZ genes were examined across various tissues and under the influence of salt stress conditions, revealing tissue-specific expression and regulation by salt stress. Through RT‒qPCR experiments, it was discovered that the relative expression levels of these six MsJAZ genes increased under salt stress. Conclusions: In summary, our study represents the first comprehensive identification and analysis of the JAZ gene family in alfalfa. These results provide important information for exploring the mechanism of JAZ genes in alfalfa salt tolerance and identifying candidate genes for improving the salt tolerance of autotetraploid cultivated alfalfa via genetic engineering in the future. [ABSTRACT FROM AUTHOR]

SUMMARY: Drought is a major environmental factor that limits the production of alfalfa (Medicago sativa). In the present study, M. sativa NUCLEAR TRANSPORT FACTOR 2‐LIKE (MsNTF2L) was identified as a nucleus‐, cytoplasm‐, and plasma membrane‐localized protein. Its transcriptional expression was highly induced by ABA and drought stress. Overexpression of MsNTF2L in Arabidopsis resulted in hypersensitivity to ABA during both the seed germination and seedling growth stages. However, transgenic Arabidopsis plants exhibited enhanced tolerance to drought stress by reducing the levels of reactive oxygen species (ROS) and increasing the expression of stress/ABA‐inducible genes. Consistently, analysis of MsNTF2L overexpression (OE) and RNA interference (RNAi) alfalfa plants revealed that MsNTF2L confers drought tolerance through promoting ROS scavenging, a decrease in stomatal density, ABA‐induced stomatal closure, and epicuticular wax crystal accumulation. MsNTF2L highly affected epicuticular wax deposition, as a large group of wax biosynthesis and transport genes were influenced in the alfalfa OE and RNAi lines. Furthermore, transcript profiling of drought‐treated alfalfa WT, OE, and RNAi plants showed a differential drought response for genes related to stress/ABA signaling, antioxidant defense, and photosynthesis. Taken together, these results reveal that MsNTF2L confers drought tolerance in alfalfa via modulation of leaf water loss (by regulating both stomata and wax deposition), antioxidant defense, and photosynthesis. Significance Statement: By studying the drought‐inducible genes of alfalfa (Medicago sativa), we discovered that the nucleus‐, cytoplasm‐, and plasma membrane‐localized protein NUCLEAR TRANSPORT FACTOR 2‐LIKE improves drought tolerance by modulating leaf water loss (by regulating both stomata and wax deposition), antioxidant defense, and photosynthesis. This gene might be targeted in future breeding programs to improve drought tolerance in alfalfa. [ABSTRACT FROM AUTHOR]

Unlike many crop seeds which are large in size, forage seeds are relatively small. Moreover, some forage seeds from the same genus, or even different genera, share similar morphologies in terms of colour, size, weight and so on. Alfalfa (Medicago sativa L.) and burr medic (Medicago polymorpha L.) also share similar morphology. However, alfalfa is a perennial, cross‐pollinated herbaceous species, whereas burr medic is considered an annual, self‐pollinating plant. Therefore, a universal and rapid method of discriminating burr medic seeds from alfalfa seeds or other similar forage seeds from different plant species is needed. Previous methods of discriminating similar‐looking forage seeds have been shown to be unreliable or time consuming. Here, a universal method that combines polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis techniques was developed to discriminate alfalfa and burr medic. Based on the differences in the lengths of homologous genes caused by insertions and deletions between two species, a pair of primers was designed to discriminate the accessions of 15 alfalfa and 9 burr medic via visualizing the clearly different PCR amplicon sizes. In addition, the melting curve analysis was performed to investigate the specificity and reliability of the primer pair. The results showed two different melting peaks between the two species, further demonstrating this method can accurately and sensitively identify the two species. Thus, we believe that this method has the great potential to be extensively applied for the identification of contaminated seeds and breeding of forages. [ABSTRACT FROM AUTHOR]

The Dof transcription factor is a plant-specific transcriptional regulator that plays important roles in plant development and acts as a mediator in plant external stress responses. However, Dofs have previously been identified in several plants but not in alfalfa (Medicago sativa L.), one of the most widely cultivated forage legumes. In the present study, a total of 40 MsDof genes were identified, and the phylogenetic reconstruction, classification, conserved motifs, and expression patterns under abscisic acid (ABA), cold, heat, drought and salt stresses of these Dof genes were comprehensively analyzed. The Dof genes family in alfalfa could be classified into eight classes. Gene ontology (GO) and tissue-specific analysis indicated that most MsDof genes may be involved in biological functions during plant growth. Moreover, the expression profiles and quantitative real-time PCR analysis indicated that eight candidate abiotic tolerance genes were induced in response to four abiotic stresses. This study identified the possibility of abiotic tolerance candidate genes playing various roles in stress resistance at the whole genome level, which would provide new information on the Dof family in alfalfa. [ABSTRACT FROM AUTHOR]

Alfalfa is the most extensively cultivated forage legume worldwide. However, the molecular mechanisms underlying alfalfa responses to exogenous abscisic acid (ABA) are still unknown. In this study, the first global transcriptome profiles of alfalfa roots under ABA treatments for 1, 3 and 12 h (three biological replicates for each time point, including the control group) were constructed using a BGISEQ-500 sequencing platform. A total of 50,742 isoforms with a mean length of 2541 bp were generated, and 4944 differentially expressed isoforms (DEIs) were identified after ABA deposition. Metabolic analyses revealed that these DEIs were involved in plant hormone signal transduction, transcriptional regulation, antioxidative defense and pathogen immunity. Notably, several well characterized hormone signaling pathways, for example, the core ABA signaling pathway, was activated, while salicylic acid, jasmonate and ethylene signaling pathways were mainly suppressed by exogenous ABA. Moreover, the physiological work showed that catalase and peroxidase activity and glutathione and proline content were increased after ABA deposition, which is in accordance with the dynamic transcript profiles of the relevant genes in antioxidative defense system. These results indicate that ABA has the potential to improve abiotic stress tolerance, but that it may negatively regulate pathogen resistance in alfalfa. [ABSTRACT FROM AUTHOR]

Cultivated alfalfa (Medicago sativa L.) is one of the most important perennial legume forages in the world, and it has considerable potential as a valuable forage crop for livestock. However, the molecular mechanisms underlying alfalfa responses to cold stress are largely unknown. In this study, the transcriptome changes in alfalfa under cold stress at 4 °C for 2, 6, 24, and 48 h (three replicates for each time point) were analyzed using the high-throughput sequencing platform, BGISEQ-500, resulting in the identification of 50,809 annotated unigenes and 5283 differentially expressed genes (DEGs). Metabolic pathway enrichment analysis demonstrated that the DEGs were involved in carbohydrate metabolism, photosynthesis, plant hormone signal transduction, and the biosynthesis of amino acids. Moreover, the physiological changes of glutathione and proline content, catalase, and peroxidase activity were in accordance with dynamic transcript profiles of the relevant genes. Additionally, some transcription factors might play important roles in the alfalfa response to cold stress, as determined by the expression pattern of the related genes during 48 h of cold stress treatment. These findings provide valuable information for identifying and characterizing important components in the cold signaling network in alfalfa and enhancing the understanding of the molecular mechanisms underlying alfalfa responses to cold stress. [ABSTRACT FROM AUTHOR]

GRAS transcription factors are one of the most important plant-specific gene families and play diverse roles in plant development and abiotic stress. A total of 55 MsGRAS genes were obtained from the autotetraploid cultivated alfalfa genome of XinJiangDaYe. Phylogenetic analysis demonstrated that the MsGRAS genes were classified into 10 subgroups, including HAM, LAS, SCL4/7, DLT, SCR, SCL3, DELLA, PAT1, SHR, and LISCL. Gene structure analysis showed that 92.7% of MsGRAS genes lacked introns. Many hormone- and stress-related cis -regulatory elements have been identified in the promoter regions of MsGRAS genes. A total of 55 MsGRAS genes were unevenly mapped on 32 alfalfa chromosomes, except for MsGRAS53 and MsGRAS55. Gene duplication analysis demonstrated that segmental duplication was the key driving force for the expansion of this gene family during evolution. Expression analysis of 55 MsGRAS genes in various tissues and under drought stress showed that they were differentially expressed. RT-qPCR analysis revealed that the expression of eight selected MsGRAS genes was significantly induced by drought stress. Collectively, our study is the first to investigate GRAS genes in autotetraploid cultivated alfalfa, and these findings will further enrich our knowledge regarding GRAS transcription factor genes and establish a foundation for enhancing drought stress tolerance by genetic engineering in autotetraploid cultivated alfalfa. • A total of 55 MsGRAS genes were identified in the autotetraploid cultivated alfalfa. • Segmental duplication was the key driving force for the expansion of MsGRAS genes. • MsGRAS genes play crucial roles in development and drought stress response in alfalfa. [ABSTRACT FROM AUTHOR]