With the ever-changing environment and climate, soil salinization has become a major environmental issue. High salinity stress expands the area of crop damage and threatens both crop quality and yield[1]. Salt stress is a limiting factor in crop growth and development. Generally, plants under salt stress have a larger proportion of roots and thus favor the retention of toxic ions[2]. Additionally, salt-tolerant species accumulate Pro and GB for osmotic regulation[3], but accelerate starch consumption to cope with salt stress[4]. It is also well known that salt induces oxidative stress in plants; in response, salt-tolerant plants exhibit an upregulation of antioxidant defences[5].
Alfalfa (Medicago sativa L.) is a widely planted perennial forage crop with a well-developed root system, rich nutrition, and a certain level of stress resistance. Alfalfa can be used as a raw biological material for ethanol production and has great potential for the future energy revolution. It is also a soil and water conservation plant with important economic and ecological functions[6−8]. Although alfalfa is rich in nutrients and has high ecological adaptability, it has some limitations with respect to agricultural production. At this stage, it is important to use molecular breeding technology to breed highly resistant dominant varieties.
CYP450 monooxygenases (CYP450s) are enzymes that contain heme-thiolate domains and play important roles in plant growth, flavonoid synthesis, and other metabolic pathways[9]. CYP450s in plants constitute the largest family of enzymes related to plant metabolism, containing 127 subfamilies and accounting for approximately 1% of the total genes in the plant genome[10,11]. CYP450s have a conserved heme domain sequence, FxxGxRxCxG — usually located in the endoplasmic reticulum, mitochondria, Golgi apparatus, and other organelle membrane systems — that combines with different substrates to catalyze reactions. Based on their evolutionary relationships, plant CYP450s are divided into 11 clans (CYP51, CYP74, CYP97, CYP710, CYP711, CYP727, CYP746, CYP71, CYP72, CYP85, and CYP86); however, new families are still being discovered[9,10]. Since the discovery of CYP450s, members of the CYP450 protein family of many plants, including Arabidopsis, rice, corn, and thistle alfalfa (Medicago truncatula), have been isolated and identified[10,12]. Numerous studies have shown that CYP450s in plants participate in the synthesis of a variety of primary and secondary metabolites, such as phenylpropanes, terpenes, flavonoids, alkaloids, fatty acids, and plant hormones. CYP450s also participate in the synthesis of cell wall structural components, protection against pests and diseases, and the decomposition of toxic substances, such as herbicides and pesticides[13]. CYP51G, CYP85A, CYP90B, CYP710A, CYP724B, and CYP736A of the CYP450 family are relatively conserved in the plant kingdom, and are mainly involved in primary metabolism related to the biosynthesis of sterols, steroid hormones, saponins, phenylpropanes, and auxins, as well as in terpene metabolism[14,15]. CYP716, CYP72, CYP88, and other CYP450s play important roles in the structural diversification and functionalization of terpenoids.
In M. truncatula, CYP716A12 has a catalytic effect on β-vanilla, converting it to oleanolic acid. CYP93B10 and CYP93B11 play important roles in flavonoid synthesis, Hansen et al.[16] demonstrated that CYP716A47 regulates ginsenoside synthesis. Arabidopsis AtCYP79B2 and AtCYP79B3 catalyze the tryptophan synthesis of indole acetaldoxime, an auxin precursor, and Arabidopsis AtCYP85A2 participates in brassinosteroid synthesis. Transgenic plants with ectopic AtCYP79B2 overexpression exhibited traits such as dwarfing and sterility. Plant CYP71, CYP72, CYP76, and other subfamily members exhibit enhanced resistance to harmful foreign substances, while the overexpression of CYP71A10 in soybeans and heterologous expression of ginseng CYP736A12 in Arabidopsis enhanced plant tolerance to phenylurea herbicides. Under drought stress, the expression of the tobacco ABA hydroxylase genes CYP707A1, CYP94C1, and CYP94B3 significantly increased.
Although CYP450s represent a large gene family in plants, the functions of most CYP450s remain unidentified; additionally, few CYP450s have a high similarity in amino acid sequence. There are few studies on the role of CYP450s in plant tolerance to abiotic stresses, such as high temperatures, drought, and salt. Previous studies have shown that most expressed CYP450s contain cis-acting elements — such as the MYB-binding site, ACGT core sequence, or TGA-box — involved in plant resistance. Despite previous research on the structure and function of CYP450s, most studies on CYP450s have focused on the secondary metabolites of models and medicinal plants, and their effects on pest and disease resistance. Few studies have been conducted on the regulatory effects of CYP450s on plant resistance, especially regarding salt tolerance in forage plants. In a previous study, using transcriptomic data analysis, we found that many CYP450 genes in alfalfa responded positively to salt stress. It was thus indicated that these CYP450 genes play important roles in salt stress and regulate plant adaptability to coercion.
CYP450s play a crucial role in the regulation of flavonoid synthesis and plant growth; however, members of the CYP450 family in alfalfa have not been analyzed and identified at the genomic level. We therefore aimed to identify the MsCYP (M. sativa CYP450) genes involved in the salt stress response and quality of alfalfa via genomic sequencing, as well as analyze a CYP450 gene model, phylogenetic relationships, chromosome locations, and other structural features. The expression patterns of key CYP450 genes were analyzed using RNA-seq (RNA sequencing) and RT-qPCR (Real time quantitative PCR). This study therefore lays the foundation for the exploration of CYP450 gene function, and provides valuable information for improving alfalfa varieties under high stress.