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Flavonoids are usually present in forms of glucosides in plants, which could be catabolized by β-glucosidase (BGLU) to form their corresponding flavonoid aglycones. In this study, we isolated three abiotic-responsive BGLU genes (MtBGLU17, MtBGLU21 and MtBGLU22) from Medicago truncatula, and found only the recombinant MtBGLU17 protein could catalyse the hydrolysis of flavonoid glycosides. The recombinant MtBGLU17 protein is active towards a variety of flavonoid glucosides, including glucosides of flavones (apigenin and luteolin), flavonols (kaempferol and quercetin), isoflavones (genistein and daidzein) and flavanone (naringenin). In particular, the recombinant MtBGLU17 protein preferentially hydrolyses flavonoid-7-O-glucosides over their corresponding 3-O-glucosides. The content of luteoin-7-O-glucoside was reduced in the MtBGLU17 overexpression plants but increased in the Tnt-1 insertional mutant lines, whereas luteoin content was increased in the MtBGLU17 overexpression plants but reduced in the Tnt-1 insertional mutant lines. Under drought and salt (NaCl) treatment, the MtBGLU17 overexpression lines showed relatively higher DPPH content, and higher CAT and SOD activity than the wild type control. These results indicated that overexpression lines of MtBGLU17 possess higher antioxidant activity and thus confer drought and salt tolerance, implying MtBGLU17 could be potentially used as a candidate gene to improve plant abiotic stress tolerance., (© 2024 John Wiley & Sons Ltd.)

Nuclear factor YB (NF-YB) are plant-specific transcription factors that play a critical regulatory role in plant growth and development as well as in plant resistance against various stresses. In this study, a total of 49 NF-YB genes were identified from the genomes of Medicago truncatula and Medicago sativa . Multiple sequence alignment analysis showed that all of these NF-YB members contain DNA binding domain, NF-YA interaction domain and NF-YC interaction domain. Phylogenetic analysis suggested that these NF-YB proteins could be classified into five distinct clusters. We also analyzed the exon-intron organizations and conserved motifs of these NF-YB genes and their deduced proteins. We also found many stress-related cis -acting elements in their promoter region. In addition, analyses on genechip for M. truncatula and transcriptome data for M. sativa indicated that these NF-YB genes exhibited a distinct expression pattern in various tissues; many of these could be induced by drought and/or salt treatments. In particular, RT-qPCR analysis revealed that the expression levels of gene pairs MsNF-YB27/MtNF-YB15 and MsNF-YB28/MtNF-YB16 were significantly up-regulated under NaCl and mannitol treatments, indicating that they are most likely involved in salt and drought stress response. Taken together, our study on NF-YB family genes in Medicago is valuable for their functional characterization, as well as for the application of NF-YB genes in genetic breeding for high-yield and high-resistance alfalfa.

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 .

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. truncatula P450 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.

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 .

β-glucosidases (BGLUs) hydrolyze the β-D-glycosidic bond with retention of anomeric configuration. BGLUs were associated with many aspects of plant physiological processes, in particular biotic and abiotic stresses through the activation of phytohormones and defense compounds. However, studies on systematic analysis of the stress- or hormone-responsive BGLU genes in plant are still rare. In this study, total 51 BGLU genes of the glycoside hydrolase family 1 were identified in the genome of the model legume plant Medicago truncatula, and they were classified into five distinct clusters. Sequence alignments revealed several conserved and characteristic motifs among these MtBGLU proteins. Analyses of their putative signal peptides and N-glycosylation site suggested that the majority of MtBGLU members have dual targeting to the vacuole and/or chloroplast. Many regulatory elements possibly related with abiotic stresses and phytohormones were identified in MtBGLU genes. Moreover, Microarray and qPCR analyses showed that these MtBGLU genes exhibited distinct expression patterns in various tissues, and in response to different abiotic stress and hormonal treatments. Notably, MtBGLU21, MtBGLU22, MtBGLU28, and MtBGLU30 in cluster I were dramatically activated by NaCl, PEG, IAA, ABA, SA and GA3 treatments. Collectively, our genome-wide characterization, evolutionary analysis, and expression pattern analysis of MtBGLU genes suggested that BGLU genes play crucial roles in response to various abiotic stresses and hormonal cues in M. truncatula., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Plant β-galactosidases (BGAL) function in various cell wall biogeneses and modifications, and they belong to the glycoside hydrolase family. However, the roles of BGAL family members in Medicago truncatula cell wall remodeling remain unclear. In this study, a total of 25 MtBGAL members of the glycoside hydrolase gene family 35 were identified, and they were clustered into nine sub-families. Many cis-acting elements possibly related to MeJA and abscisic acid responses were identified in the promoter region of the MtBGAL genes. Transcript analyses showed that these MtBGAL genes exhibited distinct expression patterns in various tissues and developing stem internodes. Furthermore, a stem-specific expression module associated with cell wall metabolic pathways was identified by weighted correlation network analysis (WGCNA). In particular, MtBGAL1 and MtBGAL23 within the stem-specific expression module were highly expressed in mature stems. In addition, several genes involved in lignin, cellulose, hemicellulose and pectin pathways were co-expressed with MtBGAL1 and MtBGAL23. It was also found that MtBGAL1 and MtBGAL23 were localized to the cell wall at the subcellular level, indicating their roles in the modification of cell wall metabolites in Medicago. As a whole, these results will be useful for further functional characterization and utilization of BGAL genes in cell wall modifications aiming to improve the quality of legume forage crops. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]