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.

Growth Regulatory Factors (GRF) are plant-specific transcription factors that play critical roles in plant growth and development as well as plant tolerance against stress. In this study, a total of 16 GRF genes were identified from the genomes of Medicago truncatula and Medicago sativa. Multiple sequence alignment analysis showed that all these members contain conserved QLQ and WRC domains. Phylogenetic analysis suggested that these GRF proteins could be classified into five clusters. The GRF genes showed similar exon–intron organizations and similar architectures in their conserved motifs. Many stress-related cis-acting elements were found in their promoter region, and most of them were related to drought and defense response. In addition, analyses on microarray and transcriptome data indicated that these GRF genes exhibited distinct expression patterns in various tissues or in response to drought and salt treatments. In particular, qPCR results showed that the expression levels of gene pairs MtGRF2–MsGRF2 and MtGRF6–MsGRF6 were significantly increased under NaCl and mannitol treatments, indicating that they are most likely involved in salt and drought stress tolerance. Collectively, our study is valuable for further investigation on the function of GRF genes in Medicago and for the exploration of GRF genes in the molecular breeding of highly resistant M. sativa.

The plant respiratory burst oxidase homologs (RBOHs) are crucial enzymes responsible for the production of reactive oxygen species (ROS) in plants, playing a pivotal role in regulating various aspects of plant growth, development, and stress responses. While RBOH family members have been identified across a wide range of plant species, the functions and characteristics of the RBOH gene family in oats remain poorly understood. In this study, 35 members of the RBOH gene family in the oat genome were identified using bioinformatics approaches. Conserved motif and gene structure analyses revealed that most AsRBOH genes contain Motif4 and Motif5. Phylogenetic tree analysis demonstrated that the AsRBOHs can be classified into five distinct subfamilies. Synteny analysis indicated that AsRBOHs share the highest number of syntenic gene pairs with wheat. Additionally, cis-regulatory element analysis identified several elements associated with drought and hypoxia-specific responses in AsRBOHs. Expression analysis using qRT-PCR showed that 28 AsRBOH genes were upregulated under drought stress, while 18 were downregulated under salt stress. Notably, the genes 7DG1382190 and 7AG1225850 were found to be involved in both drought and salt stress responses. In conclusion, these findings provide a valuable foundation for future functional studies of the AsRBOH gene family in oats, offering insights that could contribute to the improvement and innovation of oat varieties and germplasm.

Epidermal pattern factor-like (EPF/EPFL) genes are a unique class of small, secreted peptides found in plants that play crucial roles in plant stress responses. A genome-wide analysis revealed 33 AsEPF/EPFL genes in oats (Avena sativa), with 28 containing the conserved EPF domain and 5 harbouring the stomagen domain. These proteins share 2–6 conserved motifs, reflecting functional modularity. The phylogenetic classification grouped these genes into five evolutionarily conserved clades containing both monocot and dicot homologues, indicating early divergence prior to monocot–dicot speciation. Expression profiling revealed distinct tissue-specific patterns: preferential expression in roots (12 genes), stems (6 genes), leaves (5 genes), and spikes (7 genes), with 3 genes showing dual peak expression in stems and leaves. Further analysis of gene expression under salt and drought stress revealed that AsEPF/EPFLs are induced by both types of stress, with different genes showing varying expression patterns under drought and salt stress. This study identified valuable candidate genes for high-yielding and stress-resistant oat breeding.