Alfalfa (Medicago sativa L.) is considered to be the most important forage crop on a global scale. Nevertheless, soil salinity significantly decreases productivity, seriously threatening food security worldwide. One viable strategy is to explore salt stress‐responsive factors and elucidate their underlying molecular mechanism, and utilize them in further alfalfa breeding. In the present study, we designated MsWRKY33 as a representative salt stress‐responsive factor preferentially expressed in alfalfa roots and leaves. Subsequently, it was demonstrated that MsWRKY33 was localized in the cell nucleus, and functioned as a transcriptional activator of the W‐box element. Transgenic alfalfa overexpressing MsWRKY33 displayed enhanced salt stress tolerance and antioxidant activities with no significant difference in other agronomic traits. Transcriptome profiling of MsWRKY33 transgenic alfalfa under control and salt treatment unveiled significantly altered expression of reactive oxygen species (ROS) scavenger genes in transgenic alfalfa. Subsequent examination revealed that MsWRKY33 binded to the promoter of MsERF5, activating its expression and consequently fine‐tuning the ROS‐scavenging enzyme activity. Furthermore, MsWRKY33 interacted with the functional fragment of MsCaMBP25, which participates in Ca²⁺ signaling transduction. Collectively, this research offers new insight into the molecular mechanism of alfalfa salt stress tolerance and highlights the potential utility of MsWRKY33 in alfalfa breeding.

The calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) play important roles in plant signal transduction and response to abiotic stress. Plants of Medicago genus contain many important forages, and their growth is often affected by a variety of abiotic stresses. However, studies on the CBL and CIPK family member and their function are rare in Medicago. In this study, a total of 23 CBL and 58 CIPK genes were identified from the genome of Medicago sativa as an important forage crop, and Medicaog truncatula as the model plant. Phylogenetic analysis suggested that these CBL and CIPK genes could be classified into five and seven groups, respectively. Moreover, these genes/proteins showed diverse exon-intron organizations, architectures of conserved protein motifs. Many stress-related cis-acting elements were found in their promoter region. In addition, transcriptional analyses showed that these CBL and CIPK genes exhibited distinct expression patterns in various tissues, and in response to drought, salt, and abscisic acid treatments. In particular, the expression levels of MtCIPK2 (MsCIPK3), MtCIPK17 (MsCIPK11), and MtCIPK18 (MsCIPK12) were significantly increased under PEG, NaCl, and ABA treatments. Collectively, our study suggested that CBL and CIPK genes play crucial roles in response to various abiotic stresses in Medicago.

Cytochrome P450 monooxygenases (P450s) catalyze a great number of biochemical reactions and play vital roles in plant growth, development and secondary metabolism. As yet, the genome-scale investigation on P450s is still lacking in the model legume Medicago truncatula. In particular, whether and how many MtP450s are involved in drought and salt stresses for Medicago growth, development and yield remain unclear. In this study, a total of 346 MtP450 genes were identified and classified into 10 clans containing 48 families. Among them, sixty-one MtP450 genes pairs are tandem duplication events and 10 MtP450 genes are segmental duplication events. MtP450 genes within one family exhibit high conservation and specificity in intron–exon structure. Meanwhile, many Mt450 genes displayed tissue-specific expression pattern in various tissues. Specifically, the expression pattern of 204 Mt450 genes under drought/NaCl treatments were analyzed by using the weighted correlation network analysis (WGCNA). Among them, eight genes (CYP72A59v1, CYP74B4, CYP71AU56, CYP81E9, CYP71A31, CYP704G6, CYP76Y14, and CYP78A126), and six genes (CYP83D3, CYP76F70, CYP72A66, CYP76E1, CYP74C12, and CYP94A52) were found to be hub genes under drought/NaCl treatments, respectively. The expression levels of these selected hub genes could be induced, respectively, by drought/NaCl treatments, as validated by qPCR analyses, and most of these genes are involved in the secondary metabolism and fatty acid pathways. The genome-wide identification and co-expression analyses of M. truncatulaP450 superfamily genes established a gene atlas for a deep and systematic investigation of P450 genes in M. truncatula, and the selected drought-/salt-responsive genes could be utilized for further functional characterization and molecular breeding for resistance in legume crops.

Background: Oat (Avena sativa L.) belongs to the early maturity grass subfamily of the Gramineae subfamily oats (Avena) and has excellent characteristics, such as tolerance to barrenness, salt, cold, and drought. Aquaporin (AQP) proteins belong to the major intrinsic protein (MIP) superfamily, are widely involved in plant growth and development, and play an important role in abiotic stress responses. To date, previous studies have not identified or analyzed the AsAQP gene family system, and functional studies of oat AQP genes in response to drought, cold, and salt stress have not been performed.Methods: In this study, AQP genes (AsAQP) were identified from the oat genome, and various bioinformatics data on the AQP gene family, gene structure, gene replication, promoters and regulatory networks were analyzed. Quantitative real-time PCR technology was used to verify the expression patterns of the AQP gene family in different oat tissues under different abiotic stresses.Results: In this study, a total of 45 AQP genes (AsAQP) were identified from the oat reference genome. According to a phylogenetic analysis, 45 AsAQP were divided into 4 subfamilies (PIP, SIP, NIP, and TIP). Among the 45 AsAQP, 23 proteins had interactions, and among these, 5AG0000633.1 had the largest number of interacting proteins. The 20 AsAQP genes were expressed in all tissues, and their expression varied greatly among different tissues and organs. All 20 AsAQP genes responded to salt, drought and cold stress. The NIP subfamily 6Ag0000836.1 gene was significantly upregulated under different abiotic stresses and could be further verified as a key candidate gene.Conclusion: The findings of this study provide a comprehensive list of members and their sequence characteristics of the AsAQP protein family, laying a solid theoretical foundation for further functional analysis of AsAQP in oats. This research also offers valuable reference for the creation of stress-tolerant oat varieties through genetic engineering techniques.

The control of flowering time has an important impact on biomass and the environmental adaption of legumes. The CCT (CO, COL and TOC1) gene family was elucidated to participate in the molecular regulation of flowering in plants. We identified 36 CCT genes in the M. truncatula genome and they were classified into three distinct subfamilies, PRR (7), COL (11) and CMF (18). Synteny and phylogenetic analyses revealed that CCT genes occurred before the differentiation of monocot and dicot, and CCT orthologous genes might have diversified among plants. The diverse spatial-temporal expression profiles indicated that MtCCT genes could be key regulators in flowering time, as well as in the development of seeds and nodules in M. truncatula. Notably, 22 MtCCT genes with typical circadian rhythmic variations suggested their different responses to light. The response to various hormones of MtCCT genes demonstrated that they participate in plant growth and development via varied hormones dependent pathways. Moreover, six MtCCT genes were dramatically induced by salinity and dehydration treatments, illustrating their vital roles in the prevention of abiotic injury. Collectively, our study provides valuable information for the in-depth investigation of the molecular mechanism of flowering time in M. truncatula, and it also provides candidate genes for alfalfa molecular breeding with ideal flowering time.

The trihelix transcription factor (GT) family is widely involved in regulating plant growth and development, and most importantly, responding to various abiotic stresses. Our study first reported the genome-wide identification and analysis of GT family genes in Medicago truncatula. Overall, 38 trihelix genes were identified in the M. truncatula genome and were classified into five subfamilies (GT-1, GT-2, SH4, GTγ and SIP1). We systematically analyzed the phylogenetic relationship, chromosomal distribution, tandem and segmental duplication events, gene structures and conserved motifs of MtGTs. Syntenic analysis revealed that trihelix family genes in M. truncatula had the most collinearity relationship with those in soybean followed by alfalfa, but very little collinearity with those in the maize and rice. Additionally, tissue-specific expression analysis of trihelix family genes suggested that they played various roles in the growth and development of specific tissues in M. truncatula. Moreover, the expression of some MtGT genes, such as MtGT19, MtGT20, MtGT22, and MtGT33, was dramatically induced by drought, salt, and ABA treatments, illustrating their vital roles in response to abiotic stresses. These findings are helpful for improving the comprehensive understanding of trihelix family; additionally, the study provides candidate genes for achieving the genetic improvement of stress resistance in legumes.