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Genome-wide identification of the cytochrome P450 family and analysis of CYP regarding salt tolerance in Medicago sativa L.

  • # These authors contributed equally: Xinyu Zhang, Li Xue

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  • With the ever-changing environment and climate, high salinity has become a major environmental issue that threatens crop quality and yield. Alfalfa (Medicago sativa L.) is a perennial forage crop planted worldwide that has a well-developed root system and salt tolerance. Cytochrome P450 monooxygenase (CYP450) genes play important roles in flavonoid synthesis, plant growth, and development. However, few studies have focused on CYP450s in forage grass, especially the role of CYP450 genes in plant resistance to environmental stresses, such as drought and high salinity. In this study, 376 menbers in MsCYP family genes were identified using hmmsearch and BLASTP in the alfalfa protein database using the AtCYP450 protein sequence. Then by exploring MsCYP gene structures, tandem and segmental duplication events, and evolutionary relationships with CYP450s in other plants, potential MsCYPs that respond to salt stress were screened. Candidate genes were selected for transient expression in tobacco and heterologous overexpression in Arabidopsis for salinity response. This study provides a foundation for verifying the function of MsCYPs in improving the quality of agricultural products.
  • Huangqin tea (HQT, Fig. 1), also called Huangjin tea in Chinese, has a long history of consumption in China[1]. It is a healthy herbal tea crafted from the aerial parts of Scutellaria baicalensis Georgi, S. scordifolia Fisch, S. amoena C. H. Wrigh, and S. viscidula Bung[24]. Unlike traditional tea made from Camellia sinensis (L.) O. Kuntze, HQT lacks stimulating components and is renowned for clearing heat and dry dampness, eliminating toxins, promoting digestion, and soothing fire[1,5]. It can be enjoyed both as a hot beverage and as a cold drink, and modern research suggests that HQT exhibits several pharmacological properties, such as anti-inflammatory, chemopreventive effects of colorectal cancer[6], anti-aging[7], cardiovascular protection, hypoglycemic effect, hypolipidemic effect, anti-tumor, anti-bacterial, anti-influenza virus, and enhance human resistance[812]. The predominant compounds found in HQT are flavonoids and essential oils and its potential health benefits are likely attributed to the presence of distinctive flavonoids, including isocarthamidin-7-O-β-D-glucuronide, carthamidin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucopyranside, chrysin-7-O-β-D-glucuronide, scutellarin, baicalin, wogonoside, and chrysin[1318].

    Figure 1.  The aerial parts of S. baicalensis Georgi, dried and brewed Huangqin tea.

    HQT is made using simple ingredients and has a straightforward preparation method. Traditionally, the above-ground portions of Scutellaria species have been utilized for crafting HQT, with the differentiation between stems and leaves remaining unessential. During the hot summer season (July to August), the aerial parts (stem, leaves, flowers) of these species of Scutellaria from a single source are collected and cut into small sections, which are then directly dried for later use. Alternatively, freshly harvested branch and leaf sections may undergo a transformative process through multiple rounds of steaming and subsequent drying within a steamer. Once suitably conditioned, they find their place within sealed containers, poised for extended storage periods. The brewing ritual commences by incorporating 7−8 g of this tea into 2 L of water, with subsequent replenishments of water at intervals 2−3 times. As the brewing ritual concludes, HQT graces the senses with a resplendent golden infusion—earning it another name, 'Huangjin tea'.

    HQT boasts a range of health advantages and is frequently gathered from the upper sections of Scutellaria species plants cultivated in the vicinity, intended for tea consumption. In recent years, large planting bases have been established in several regions, such as Beijing, Inner Mongolia Wuchuan, Yakeshi, Hebei Chengde, Shanxi Province, and Shandong Province, to comprehensively develop S. baicalensis resources. HQT has gained more attention and is now produced by specialized operating companies in various grades, such as bulk tea, bagged tea, and unique tea culture[12].

    To provide up-to-date information on the chemical composition, bioactivity, and safety aspects of the Scutellaria genus from HQT, this review paper has collected related literature from various databases, such as PubMed, Web of Science, Sci Finder, Scopus, Baidu Scholar, China National Knowledge Internet (CNKI), Wanfang and Weipu Data. This paper aims to draw attention to the need for further research and application of HQT in preventing and managing certain chronic diseases.

    HQT can be derived from several Scutellaria species. S. baicalensis is the most extensively cultivated, with a significant biomass in its aerial parts. Consequently, the primary source of HQT is the aerial part of S. baicalensis, and research focused on these aerial parts is also the most extensive.

    The aerial part and the root of S. baicalensis share similarities in their primary constituents, notably the significant presence of flavonoids, which are regarded as the main active components. However, there are specific chemical differences between the above-ground and underground parts of S. baicalensis. Our preliminary experiments have revealed the disparities in chemical composition between these two parts[18]. Notably, the root of S. baicalensis finds its primary use in medicinal applications, characterized by notably higher expression levels of specific 4′-deoxyflavones compared to the aerial organs. In contrast, the above-ground parts of the plant are employed to prepare tea. This differentiation in utilizing these plant components reflects the accumulated wisdom of generations cultivated through extensive periods of tasting and experience.

    This section provided an in-depth review of the chemical composition of the plant from which HQT is derived. Additionally, a specific emphasis on reviewing the aerial parts of S. baicalensis, which serve as the primary source of HQT has been placed.

    In the 1970s, researchers began to study the chemical composition of the aerial parts of S. baicalensis. The flavonoids are the most abundant chemical components in the aerial parts (flowers, stems, and leaves), and about 54 flavonoid compounds were identified (Table 1, Fig. 2). Most of them are flavonoids, dihydro flavonoids, and glycosides. The main glycoside-forming sugars are arabinose, glucose, and glucuronide, with the most abundant glucuronide glycosides. The optimization of extraction processes can yield a remarkable total flavonoid content of up to 5% in the stems and leaves of S. baicalensis[19].

    Table 1.  Flavonoids of HQT.
    NumberNameFormulaSpeciesRef.
    12',5-Dihydroxy-3',6,7,8-tetramethoxyflavoneC19H18O8S. baicalensis[17]
    22',6-DihydroxyflavoneC15H10O4S. baicalensis[16]
    33',4',5,5',7-PentamethoxyflavoneC20H20O7S. baicalensis[16]
    44',5-Dihydroxy-3',5',6,7-tetramethoxyflavoneC19H18O8S. baicalensis[22,27]
    54',5-Dihydroxy-7-methoxyflavanoneC16H14O5S. baicalensis[16]
    65,4′-Dihydroxy-6,7,8,3′-tetramethoxyflavoneC19H18O8S. baicalensis[17]
    75,2′-Dihydroxy-6,7,8-trimethoxyflavoneC18H16O7S. baicalensis[17]
    85,2′-Dihydroxy-6,7,8,3′-tetramethoxyflavoneC19H18O8S. baicalensis[17]
    95,2′-Dihydroxy-7,8-dimethoxyflavoneC17H14O6S. baicalensis[17]
    105,2′-Dihydroxy-7,8,6′-trimethoxyflavoneC18H16O7S. baicalensis[17]
    115,6,7,3',4'-Pentahydroxyflavone-7-O-glucuronideC21H20O13S. baicalensis[13]
    125,6,7,4'-TetrahydroxydihydroflavoneC15H12O6S. baicalensis[17]
    135,6,7-Trihydroxy-4'-methoxyflavoneC16H12O6S. baicalensis[15]
    145,7-Dihydroxy-6-methoxyflavanonC16H14O5S. baicalensis[17]
    155,7,4′-Trihydroxy-6-methoxyflavanoneC16H14O6S. baicalensis[22]
    165,7,4'-TrihydroxyflavanoneC15H12O6S. baicalensis[13]
    17(2S)-5,7,8,4'-Tetrahydroxyflavanone 7-O-β-D-glucuronopyranosideC21H20O12S. baicalensis[21]
    18(2S)-5,6,7,4'-Tetrahydroxyflavanone 7-O-β-D-glucuronopyranosideC21H20O12S. baicalensis[21]
    196-Hydroxyluteolin-7-O-glucuronideC21H18O13S. baicalensis[13]
    207-MethoxychrysinC16H14O4S. baicalensis[16]
    21ApigeninC15H10O5S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [17,2125]
    22Apigenin-4'-glucopyransideC21H20O10S. baicalensis[17]
    23Apigenin-6-C-glucoside-8-C-arabinosideC26H28O14S. baicalensis[13]
    24Apigenin-7-O-β-D-glucopyransideC21H20O10S. baicalensis[17]
    25Apigenin-7-O-β-D-glucuronideC21H18O11S. baicalensis, S. amoena,
    S. scordifolia
    [13,23,24]
    26Apigenin-7-O-methylglucuronideC22H20O11S. baicalensis[28]
    27BaicaleinC15H10O5S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [2225]
    28Baicalein-7-O-D-glucopyransideC21H20O10S. baicalensis[22]
    29BaicalinC21H18O11S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [16,2325]
    30CarthamidinC15H12O6S. baicalensis[13,20]
    31Carthamidin-7-O-β-D-glucuronideC21H20O12S. baicalensis[22]
    32ChrysinC15H10O4S. baicalensis, S. amoena,
    S. scordifolia
    [13,2124]
    33Chrysin-7-O-β-D-glucuronideC21H20O9S. baicalensis, S. amoena[23,29]
    34DihydrobaicalinC21H20O11S. baicalensis[22,28]
    35Dihydrooroxylin AC21H20O11S. baicalensis[13]
    36GenkwaninC16H12O5S. baicalensis[16]
    37IsocarthamidinC15H10O6S. baicalensis[20,28]
    38Isocarthamidin-7-O-β-D-glucuronideC21H20O12S. baicalensis[28]
    39IsoschaftsideC26H28O14S. baicalensis[13,16]
    40IsoscutellareinC15H10O6S. baicalensis[21,30]
    41Isoscutellarein 8-O-β-D-glucuronideC21H18O12S. baicalensis[21]
    42Kaempferol 3-O-β-D-glucopyranosideC21H20O11S. baicalensis[13,28]
    43LuteolinC15H10O6S. baicalensis[22,30]
    44Norwogonin-7-O-glucuronideC21H18O12S. baicalensis, S. amoena[13,17,23]
    45Oroxylin AC16H12O5S. baicalensis, S. amoena[17,23]
    46Oroxylin A-7-O-D-glucopyransideC22H22O10S. baicalensis[22,28]
    47Oroxylin A-7-O-β-D-glucuronideC22H20O11S. baicalensis, S. amoena[13,23]
    48PinocembrinC16H12O5S. baicalensis[13]
    49Pinocembrin-7-O-glucuronideC21H20O11S. baicalensis[13,16,22]
    50SalvigeninC18H16O6S. baicalensis[21]
    51ScutellareinC15H10O6S. baicalensis[28]
    52ScutellarinC21H18O12S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [2325,29]
    53WogoninC16H12O5S. baicalensis, S. amoena,
    S. scordifolia, S. viscidula
    [2125]
    54WogonosideC22H20O11S. baicalensis, S. viscidula[13,25]
     | Show Table
    DownLoad: CSV
    Figure 2.  Chemical structure of flavonoids in HQT.

    In 1976, Takido et al.[20] isolated two flavanone derivatives, carthamidin and isocarthamidin, for the first time as natural products from the leaves of S. baicalensis. Later, Yukinori et al.[21] identified two new flavanones, (2S)-5,7,8,4'-tetrahydroxyflavanone 7-O-β-D-glucuronopyranoside and (2S)-5,6,7,4'-tetrahydroxyflavanone 7-O-β-D-glucuronopyranoside), in the leaves of S. baicalensis. Eight compounds of chrysin, wogonin, apigenin, salvigenin, scutellarein, isoscutellarein, apigenin 7-O-glucuronide, and isoscutellarein 8-O-glucuronide were also isolated. Wang et al.[15] used column chromatography to isolate seven flavonoids (wogonin, chrysin, 5,6,7-trihydroxy-4'-methoxyflavone, carthamindin, isocarthamidin, scutellarein, and chrysin 7-O-β-D-glucuronide) from a water extract of the leaves of S. baicalensis. Liu et al.[13] identified 21 flavonoids in the stems and leaves of S. baicalensis by HPLC-UV/MS and NMR, and found one flavonone (5,6,7,3',4'-Pentahydroxyflavone-7-O-glucuronide) was a new compound. Zhao[17] firstly isolated 5,6,7,4′-tetrahydroxyflavanone 7,5,7-dihydroxy-6-methoxyflavanone, oroxylin A, 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone, 5,2′-dihydroxy-6,7,8,3′-tetramethoxyflavone, 5,2′-dihydroxy-7,8,6′-trimethoxyflavone, 5,2′-dihydroxy-7,8-dimethoxyflavone, 5,2′-dihydroxy-6,7,8-trimethoxyflavone, apigenin 4'-β-D-glucopyranoside, and apigenin-7-β-D-glucopyranoside from the aerial parts of S. baicalensis. Ma[22] firstly isolated 5,7,4'-trihydroxy-6-methoxyflavone, 5,4'-dihydroxy-6,7,3',5'-tetramethoxyflavone, from stems and leaves of S. baicalensis. Wang et al.[16] isolated 5,4'-dihydroxy-7-methoxyflavanone, genkwanin, 7-methoxychrysin, 3',4',5,5',7-pentamethoxyflavone from 60% ethanol extracts for stems and leaves of S. baicalensis for the first time. Also, the compounds of carthamidin-7-O-β-D-glucuronide, oroxylin A-7-O-β-D--glucuronide, and chrysin were isolated from this plant for the first time.

    The concentration of these chemical components in HQT varies depending on the plant part utilized. Employing the HPLC-DAD method, Shen et al.[18] established that the aerial parts (stems, leaves, and flowers) of S. baicalensis are rich in flavonoids, resembling the roots in composition but exhibiting significant disparities in content. The contents of isocarthamidin-7-O-β-D-glucuronide (106.66 ± 22.68 mg/g), carthamidin-7-O-β-D-glucuronide (19.82 ± 11.17 mg/g), and isoscutellarein-8-O-β-D-glucuronide (3.10 ±1.73 mg/g) were the highest in leaves. The content of apigenin-7-O-β-D-glucopyranoside (18.1 ± 4.85 mg/g) and chrysin-7-O-β-D-glucuronide (9.82 ± 5.51 mg/g) were the highest in flowers. HQT has a high content proportion of flavone glycosides, which is closely related to the activity of HQT. The concentrations of the nine main flavonoids in HQT infusions were measured using HPLC. The content of isocarthamidin-7-O-β-D-glucuronide (52.19 ± 29.81 mg/g) was the highest; carthamidin-7-O-β-D-glucuronide (31.48 ± 6.82 mg/g), chrysin-7-O-β-D-glucuronide (10.65 ± 0.40 mg/g) and apigenin-7-O-β-D-glucopyranside (5.39 ± 0.92 mg/g) were found at moderate levels in HQT samples. As for flavone aglycones, scutellarin (12.77 ± 1.14 mg/g), baicalin (1.88 ± 0.48 mg/g), isoscutellarein-8-O-β-D-glucuronide (2.84 ± 0.60 mg/g), wogonoside (0.23 ± 0.02 mg/g) and chrysin (0.03 ± 0.01 mg/g) has lower content in HQT[6].

    Although there are few studies on the chemical constituents of the aerial parts of S. amoena, S. scordifolia, and S. viscidula it has been shown that the compounds of the aerial parts are similar to S. baicalensis. The aerial parts of S. amoena contain the compounds of baicalein, baicalin, oroxylin A, oroxylin A-7-O-β-D-glucuronide, wogonin, chrysin, chrysin-7-O-β-D-glucuronide, norwogonin, 5,7-dihydroxy-6,8-dimethoxyflavone, scutellarin[23]. Zhang et al.[24] identified compounds of chrysin, wogonin, baicalein, apigenin, apigenin-7-O-β-D-glucoside, baicalin, and scutellarin in whole plants of S. scordifolia. The stems and leaves of S. viscidula all contain compounds of wogonoside, apigenin, baicalein, wogonin, baicalin, and scutellarin. The contents of baicalein, wogonoside, wogonin, and apigenin in the stem of S. viscidula were higher than those in the stem of S. baicalensis. In the leaves of the two species, the content of scutellarin was higher, while the content of other compounds was lower[25]. The content of scutellarin in S. viscidula was stem (2.30%) > leaf (1.78%) > flowers (0.38%)[26].

    The aerial parts of S. baicalensis are rich in essential oils, and the taste of HQT is closely related to this. The flowers of S. baicalensis are thought to have a Concord grape aroma, while HQT has a bitter flavor with distinctive herbal notes. Extensive analysis has identified 145 components in the essential oil obtained from the aerial parts of S. baicalensis. These components span various chemical classes, such as alkanes, carboxylic acids, fatty acids, monoterpenes/oxygenated monoterpenes, sesquiterpenes triterpenoids and Vitamins (Supplemental Table S1), which have demonstrated their efficacy in combatting bacteria, reducing inflammation and inhibiting tumor growth[2731]. Among these, major constituents include germacrene D (5.4%−39.3%), β-caryophyllene (29.0%), caryophyllene (18.9%), eugenol (18.4%), caryophyllene (15.2%), caryophyllene oxide (13.9%), (E)-β-caryophyllene (11.6%), 5-en-3-stigmasterol (11.3%), carvacrol (9.3%), thymol (7.5%), vitamin E (7.4%), neophytadiene (7.3%), γ-elemene (6.2%), 1-octen-3-ol (6.1%), allyl alcohol (5.5%), bicyclogermacrene (4.8%), myristicin (4.7%), acetophenone (4.6%), α-amyrin (4.6%), β-amyrin (4.4%), germacrene d-4-ol (4.3%), spathulenol (4.2%), β-pinene (4.1%), α-humulen (4.0%), 1-vinyl-1-methyl-2-(1-methylvinyl)-4-(1-methylethylidene)-cyclohexane (4.0%) are found in the aerial parts of S. baicalensis from different places[2731].

    Takeoka et al.[27] identified 64 components in volatile components of S. baicalensis flowers by solid-phase microextraction and analyzed them by GC and GC-MS. These flowers were collected at San Francisco State University (USA). Among the flower volatiles, the content of β-caryophyllene, germacrene D, δ-cadinene, γ-muurolene, and γ-cadinene were more than 3%. The essential oil obtained from the stem of S. baicalensis is mainly composed of diphenylamine, 2,2-methylenebis (6-tert-butyl-4-methylphenol), bornyl acetate, β-caryophyllene, germacrene D and 1-octen-3-ol.[32]. Gong et al.[28] analyzed and identified the specific chemical constituents of the aerial parts of S. baicalensis by using GC-MS technology and identified 37 compounds in total, such as allyl alcohol, acetophenone, caryophyllene, α-humulene, germacrene D, and γ-elemene. The plant material was collected in the Qinling Mountains in China. Lu et al.[29] found a big difference in essential oil components between the aerial and root of S. baicalensis from Kunming Botanical Garden, Yunnan Province (China). The aerial part of S. baicalensis mainly contained enols and sterols such as neophytadiene and vitamin E. However, it has the same compounds as the roots, such as nerolidol, hexadecanoic acid, 1,2-benzenedicarboxylic acid, squalene, stigmast-4-en-3-one, and partial alkanes. Recently, Wang et al.[31] found the essential oil level of the aerial parts of S. baicalensis was 0.09% (v/w, based on fresh weight) while its density was 0.93 g/mL, and obtained 31 components accounting for 97.64% of the crude essential oil, including sesquiterpenoid, monoterpenoids, phenylpropanoids, and others. It is also reported that the major components of the essential oil from the aerial parts of S. baicalensis were myristicin, eugenol, caryophyllene, caryophyllene oxide, germacrene D, spathulenol, and β-pinene, with eugenol as the most abundant. The sample of the aerial parts were harvested from Tangshan City (China). The composition of S. baicalensis essential oils varies according to the plant part used, geographical location, and growing conditions.

    Zgórka & Hajnos[33] identified the phenolic acid compounds of aerial parts of S. baicalensis by solid-phase extraction and high-speed countercurrent chromatography: p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, and caffeic acid. Chirikova & Olennikov[34] found that the aerial part of S. baicalensis contains 11 kinds of saturated fatty acids and nine kinds of unsaturated fatty acids, among which the palmitic acid content is the highest. Chlorogenic acid, fernlic acid, protocatechuic acid, vanillic acid, rosmarinic acid, caffeic acid, p-hydroxybenzoic acid, and p-coumaric acid were also detected.

    Zhao [17] isolated four sterol compounds: β-sitosterol-3-O-β-D-glucoside, α-apinasterol, β-sitosterol, and four ester compounds: methoxyphaeophorbide, p-hydroxybenethyl ethanol hexadecanoic methyl ester, ethoxyphaeophorbide, and n-octadecanol, lutein from the aerial parts of S. baicalensis.

    It is reported that flavonoids and diterpenes are the two main groups of active constituents in the genus Scutellaria. However, only one diterpene (scutebaicalin) was identified in the stems and leaves of S. baicalensis[35].

    By atomic absorption spectrophotometry, Yuan et al.[36] determined the contents of 11 metal elements in different parts of S. baicalensis. It was found that the leaves and stems of S. baicalensis were rich in Mg, K, Cr, Ni, Co, Fe, Mn, and Pb. Meanwhile, Yan et al.[37] developed an inductively coupled plasma mass spectrometry method and determined 23 kinds of inorganic elements in the stems and leaves of S. baicalensis from eight regions. Although there were no differences in the types of inorganic elements in the stems and leaves of S. baicalensis from the different areas, the content of these elements varied significantly. Among these elements, Fe, Zn, Cu, Mn, Cr, Co, Ni, Sr, B, and Ni were essential human body elements. The content of Al (516.83 μg/g) and Fe (700.62 μg/g) was the highest, while the content of B (31.54 μg/g), Ti (23.10 μg/g), Mn (65.64 μg/g), Sr (62.27 μg/g), and Ba (89.68 μg/g) was relatively high.

    Olennikov et al.[38] studied the water-soluble polysaccharides from the aerial parts of S. baicalensis from Russia and found that the polysaccharides from S. baicalensis gradually accumulated before flowering and progressively decreased after flowering.

    Yan et al.[39] found that the stems and leaves of S. baicalensis were rich in amino acids, and there was no difference in the kinds of amino acids among different producing areas, but there was a significant difference in the contents of amino acids. The content of proline, threonine, glutamic acid, lysine, glutamine, and arginine was higher, and the content of methionine, hydroxyproline, and citrulline was low.

    Several studies have focused on the functional properties of HQT, with increasing attention given to the aerial parts of S. baicalensis as the main raw material for HQT production. S. baicalensis stems and leaves flavonoids (SSF) are considered the functional components of HQT. Modern pharmacology has shown that the flavonoids extracted from the stem and leaf of S. baicalensis have been found to possess anti-inflammatory, anti-bacterial, antiviral, antipyretic and analgesic, anti-tumor, hepatoprotective, antioxidant, hypoglycemic, hypolipidemic, detoxification, myocardial ischemia protection, brain injury protection, and immunomodulatory effects. However, few studies have been conducted on the individual flavonoid compounds in the total flavonoid extract from S. baicalensis. Therefore, this paper aims to summarize and supplement the current research on the functional properties of HQT and its primary raw material (S. baicalensis) extract.

    Injury and infection could lead to inflammation, which plays a key role in the accelerated pathogenesis of immune-mediated disease[40]. Tong et al.[41], Zhou et al.[42], and Zhao et al.[43] found that S. baicalensis stem-leaf total flavonoid (SSTF) could inhibit acute exudative inflammation caused by xylene, glacial acetic acid, and egg white and also have a significant inhibitory effect on chronic inflammatory of granulation tissue hyperplasia. Wang et al.[44] observed the effect of SSTF on the aerocyst synovitis of the rat model and found that it could reduce capillary permeability, reduce the aggregation of neutrophils and basophils in tissues, reduce histamine, bradykinin, and other substances that increase vascular permeability, which is conducive to the recovery of vascular permeability in inflammation. Studies have shown that the SSTF significantly inhibits specific and non-specific inflammatory responses and can regulate the body's cellular and humoral immune functions. The mechanism of action is closely related to the effective reduction of capillary permeability, inhibition of PGE2 and NO synthesis in vivo, reduction of TNF-α expression, and reduction of inflammatory exudation[45,46]. SSTF (200 mg/kg) could balance the CD+4 Tlymphocyte subsets Th1/Th2 cells and the related cytokines IL-10 and IFN-γ in the rheumatoid arthritis model[47]. SSTF (17.5, 35 and 70 mg/kg for 38 d) could significantly improve the impairment of relearning ability and retention ability on memory impairment and nerve inflammation in chronic cerebral ischemia rats, which might be due to the inhibition of the proliferation of astrocyte and balanced the expression of the inflammatory factors in the brain[48]. Besides, the extract of S. baicalensis stem-leaf shows anti-inflammation effects both in vitro and in vivo. In cultured macrophage cells (RAW 264.7), the extract of S. baicalensis stem-leaf showed a strong anti-inflammation effect, which inhibited the expression of IL-1β. Similarly, it suppressed the LPS-induced transcriptional activity mediated by NF-κB in fish aquaculture[49]. S. baicalensis stems and leaves (3, 6, and 12g /kg, gavage for 7 d, once per day) have anti-inflammatory effects on 2% carrageenan-induced acute pleurisy in rats, and the mechanism of action may be related to the reduction of the production of inflammatory factors and the down-regulation of TRPV1 signaling protein[50]. The combination of S. baicalensis stems- Polygonum cuspidatum (3.5, 7, and 14 g/kg, gavage for 7 d) has a protective effect on lipopolysaccharide-induced acute lung injury rats, and its mechanism may be related to down-regulating the expression of TRPV1 and inhibiting the levels of TNF-α and IL-1β in inflammatory cells[50]. It is also reported that S. baicalensis stem-leaf combined with Morus alba (4, 8, and 16 g/kg/d, gavage, 10 d) has a protective effect on rats with acute pneumonia induced by lipopolysaccharide, and the mechanism might be related to the reduction of inflammatory factors and the down-regulation of TRPV1 signaling pathway[51]. Besides, the network pharmacology showed that S. baicalensis stem-leaf could prevent and control COVID-19 by intervening in 30 targets and 127 pathways, potentially preventing and treating inflammation caused by COVID-19[52].

    Based on the studies, S. baicalensis stem-leaf extract shows promising anti-inflammatory properties. These effects are likely mediated through a combination of factors, including the modulation of immune responses, reduction of inflammatory mediators, and potential interactions with signaling pathways like TRPV1. However, it's important to note that while these studies provide valuable insights, further research, including clinical trials, is needed to establish the full extent of its benefits and its potential for therapeutic applications in humans.

    Scutellaria baicalensis stem and leaf aqueous extract exhibit different degrees of inhibition of 36 strains from 13 kinds of bacteria, such as Staphylococcus aureus, Staphylococcus, Streptococcus pneumoniae, alpha-hemolytic streptococcus, beta-hemolytic streptococcus and Escherichia coli. This shows that anti-bacterial activity against Staphylococcus aureus is strong (MIC50 0.94 g/L, MBC 0.94 g/L). In vivo (217 mg/kg), it protects against the death of mice infected by Staphylococcus aureus and shows a certain dose-dependence[53]. Zhang et al.[54] found that the stem and leaves of S. baicalensis against Staphylococcus aureus and Shigella dysenteriae with MIC values of 1 and 4 mg/mL, respectively. Besides, it is reported that the water extract of the aerial part of S. baicalensis could inhibit the growth of several common pathogenic bacteria in aquacultures, such as Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus and V. harveyi[49].

    Zhao et al.[55] found that the active part of the stem and leaf of S. baicalensis could inhibit the cytopathic effect caused by 10 kinds of viruses such as Coxsackie B virus, influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus, and herpes simplex virus. It is suggested that the active parts of the stem and leaf of S. baicalensis can be used for the prevention and treatment of influenza virus, parainfluenza virus, coxsackievirus, and other related infectious diseases.

    These findings indicate that S. baicalensis stem and leaf extract possess anti-bacterial and antiviral properties, making it a potentially valuable natural resource for combating infections caused by various pathogens. However, while these results are promising, further research, including clinical trials, would be necessary to fully establish the effectiveness and safety of using S. baicalensis extract for preventing or treating bacterial and viral infections, including COVID-19.

    In a series of studies, Tong et al.[41] demonstrated that SSTF at a dosage of 20 mg/kg significantly reduced body temperature in rats with fever induced by subcutaneous injection of a 10% dry yeast suspension. Zhang et al.[56] conducted research on the antipyretic effect of scutellarin, an extract from S. baicalensis stems and leaves, in febrile rabbits and observed an antipyretic substantial impact induced by pyrogen. Yang et al.[57] conducted several animal experiments, where they discovered that intraperitoneal injection of effective doses of SSTF (42.2 and 84.4 mg/kg) exhibited a specific inhibitory effect on infectious fever in experimental animals. Moreover, intraperitoneal injection of appropriate SSTF doses (30.1, 60.3, and 120.6 mg/kg), as found in Yang et al.'s experiments[58], effectively inhibited the pain response in experimental animals. Furthermore, Zhao et al.[59] noted that SSTF exhibited a certain inhibitory effect on the pain response in experimental animals subjected to chemical and thermal stimulation.

    These findings suggest that S. baicalensis stem and leaf extract may have antipyretic and analgesic effects, particularly its total flavonoid component. These effects could be beneficial for managing fever and providing pain relief. However, as with any natural remedy, further research, including controlled clinical trials in humans, is necessary to fully understand the effectiveness, safety, and optimal dosing of S. baicalensis extract for these purposes.

    Amyloid protein (Aβ) has been widely recognized as the initiator of Alzheimer's disease (AD)[60]. The SSTF can improve cognitive function and delay the process of dementia. SSTF has been found to exert neuroprotective effects in AD animal models through various mechanisms. Ye et al.[61] demonstrated that oral administration of SSTF (50 mg/kg) could effectively improve cognitive function and reduce neuronal injury in Aβ25-35-3s -induced memory deficit rats. The underlying mechanisms may involve inhibiting oxidative stress and decreasing gliosis[62,63]. Furthermore, SSTF was shown to reduce Aβ-induced neuronal apoptosis by regulating apoptosis-related proteins Bax and Bcl-2[64]. Subsequent studies further validated the neuroprotective effects of SSTF. Cheng et al.[65] found that SSTF treatment inhibited neuronal apoptosis and modulated mitochondrial apoptosis pathway in composited Aβ rats. Ding & Shang[66] found that the SSF improves neuroprotection and memory impairment in rats due to its inhibition of hyperphosphorylation of multilocus Tau protein in rat brains. SSTF has also been found to exert neurogenesis-promoting effects by regulating BDNF-ERK-CREB signaling[12] and activating the PI3K-AKT-CREB pathway[67].

    In further studies, Ding et al.[11] proposed that the effect of SSF on promoting neurogenesis and improving memory impairment may be related to the regulation of abnormal expression of Grb2, SOS1, Ras, ERK, and BDNF molecules in the BDNF-ERK-CREB signaling pathway. Zhang et al.[68] found that SSF (25, 50, and 100 mg/kg) could significantly modulate okadaic-induced neuronal damage in rats, which provides a basis for evaluating SSF as a means to reduce tau hyperphosphorylation and Aβ expression in Alzheimer's disease. Cao et al.[69] found that SSTF (100 mg/kg, 60 d) may alleviate tau hyperphosphorylation-induced neurotoxicity by coordinating the activity of kinases and phosphatase after a stroke in a vascular dementia rat model. Gao et al.[70] demonstrated that the stems and leaves of S. baicalensis (SSF, 25, 50, and 100 mg/kg/d, 43 d) could inhibit the hyperphosphorylation of tau in rats' cerebral cortex and hippocampus induced by microinjection of okadaic acid, which may be related to the activities of protein kinase CDK5, PKA and GSK3β. Furthermore, Liu et al.[67] demonstrated that SSF (35, 70, and 140 mg/kg/d, 43 d) improved composited Aβ-induced memory impairment and neurogenesis disorder in rats through activated the PI3K-AKT-CREB signaling pathway and up-regulated the mRNA and protein expression of TRKB, PI3K, AKT, CREB and IGF2. More recently, a new study demonstrated that SSF (35, 70, and 140 mg/kg) alleviated myelin sheath degeneration in composited Aβ rats, potentially modulating sphingomyelin metabolism[71]. Collectively, these findings suggest that SSTF holds therapeutic potential for AD by targeting multiple Alzheimer's pathogenesis-related processes.

    Li et al.[72] confirmed that SSTF (5 mg/kg) could improve the behaviors and the numbers of dopaminergic neurons in the substantia nigra in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease in mice, and these beneficial activities appear to be associated with the reduction of the level of serum malondialdehyde.

    These studies suggest that S. baicalensis stem and leaf extract have the potential to exert neuroprotective effects in various neurodegenerative conditions, including Alzheimer's disease and Parkinson's disease. These effects could be attributed to its ability to modulate oxidative stress, apoptosis, signaling pathways, and protein hyperphosphorylation. However, as with any potential therapeutic agent, further research is needed to establish the full extent of its benefits, optimal dosages, and mechanisms of action, as well as its potential applications in human patients.

    In recent years, ischemic cerebrovascular disease has seriously threatened human health. Cerebral ischemia is one of the leading causes of death. It can occur in focal or global ischemia, with most cases associated with ischemic stroke[73]. Neuronal protection against oxidative damage has been proposed as a potential therapeutic strategy to avoid damage during ischemic stroke[74]. It is reported that SSTF can reduce neuronal apoptosis and free radical damage caused by heart and brain ischemia. Zhao et al.[75] have found that the pretreatment of SSTF (100 mg/kg/d) can protect the ischemia-reperfusion myocardium by enhancing the activity of the anti-oxidative enzyme, inhibiting lipid peroxidation and attenuating the oxygen-free radicals-mediated damage to the myocardium in rats. In further studies, Zhao et al.[76] proposed that SSTF (50, 100, or 200 mg/kg/d, 7 d) pretreatment could alleviate the neuronal damage incurred by ischemia-reperfusion, demonstrating a neuroprotective effect in focal ischemia-reperfusion rat model, which may involve the prohibition of the apoptosis of the neurons. Yu et al.[77] confirmed that SSTF (17.5, 35, and 70 mg/kg/d, 7 d) could attenuate cardiomyocyte apoptosis during ischemia reperfusion injury by down-regulating the protein expression of the JAK2 gene. Qin et al.[78] found that SSF (17.5, 35, and 70 mg/kg/d, 38 d) can decrease the expression of the NMDAR in hippocampus, and increase the expression of VEGF in the cerebral cortex of chronic cerebral ischemia rats.

    Focal cerebral ischemia-reperfusion can result in neuronal loss but strongly promotes activation and proliferation of hippocampal glial cells. Losing hippocampal neurons is considered one of the basic pathological mechanisms of cognitive impairment[79]. Zhao et al.[79] found that the pretreatment with SSTF (100 and 200 mg/kg) could improve neurological function after focal cerebral ischemia-reperfusion injury, with preventive and protective effects. Shang et al. found flavonoids from S. baicalensis (35−140 mg/kg) could attenuate neuron injury and improve learning and memory behavior in rats with cerebral ischemia/reperfusion[80]. In further studies, Kong et al.[81] found that the mechanisms of the protective effects on the brain against cerebral ischemia/reperfusion injury of SSTF may involve decreasing the content of brain water, increasing microvascular recanalization, reducing the apoptosis of hippocampal neurons, and attenuating free radical damage. Bai et al.[82] proposed that SSTF (100 mg/kg/d, 7 d) could protect the neurological function in rats following I/R injury by alleviating the damage to the ultrastructure of cerebral cortex neurons and synapse. Yan et al.[83] found that SSTF (100 mg/kg/d, 7 d) pretreatment can exert preventive, protective effects on cerebral tissue by relieving brain edema, decreasing neural damage, promoting microvascular repatency, and increasing enzyme activity. It has been reported that the SSTF may protect neurons and their synaptic structures in multiple ways, but whether this mechanism enhances the resistance of neurons to damage or increases the repair function remains to be further explored.

    Essential hypertension is a common chronic cardiovascular disease, which can lead to multiple target organ damage, such as heart, brain, and blood vessels. It is a risk factor for coronary heart disease, heart failure, and other cardiovascular diseases. It is reported that SSTF (17.5, 35.0, and 70 0 mg/kg, 8 weeks) can inhibit myocardial remodeling in primary hypertensive rats, and the medium dose exerts the best inhibitory effect, and the mechanism may be related to inflammatory response induced by inhibiting the NF-κB signaling pathway[84].

    S. baicalensis stem and leaf extract have the potential to provide neuroprotective effects in conditions related to ischemic cerebrovascular disease and hypertension. Its ability to modulate oxidative stress, inflammation, and apoptotic pathways appears to contribute to its beneficial effects. However, further research is needed to fully understand the mechanisms and optimal usage of SSTF for these therapeutic purposes.

    Aging is associated with the deterioration of physiological function and the decline of cognitive ability[85]. It is reported that the alcohol extracts from roots, stems, leaves, and flowers of S. baicalensis (400 mg/kg, 7 weeks) could regulate the content of differential metabolites in urine samples of D-gal-induced aging-model rats to different degrees and play a certain role in improving the metabolic disorders of aging rats[7]. A further study investigated the anti-aging effects and potential mechanisms of S. baicalensis leaves and flower extract. S. baicalensis leaves (400 and 800 mg/kg, 7 weeks) have an anti-aging effect, which can improve the acquired alopecia, slow response, and other characteristics of aging rats, increase the spontaneous activity of aging rats, and reduce the damage of lipid peroxidation and glycosylation induced by D-galactose[86]. The S. baicalensis flowers extract (400 and 800 mg/kg, 7 weeks) could effectively reverse the cognitive decline and oxidative stress injury and alleviate liver pathological abnormalities in the D -galactose-induced aging rats, which are involved in the glutamine-glutamate metabolic pathway[85].

    S. baicalensis extracts, particularly those from leaves and flowers, may have anti-aging properties by regulating metabolic disorders, improving cognitive function, reducing oxidative stress, and alleviating aging-related physiological abnormalities. However, further research is necessary to fully understand the mechanisms underlying these effects and to determine the potential of these extracts for human applications in addressing age-related issues.

    The SSTF (200 mg/kg d, 35 d) can reduce the joint damage of collagen-induced arthritis mice and balance the CD+4 T lymphocyte subsets Th17 and Treg cells[87]. Besides, on the multiple sclerosis model, the SSTF (100, 200 mg/kg d, 16 d) displayed a protective effect on experimental autoimmune encephalomyelitis rats through a balance of the CD+4 Tlymphocyte subsets Th17 and Treg cells[88]. Zhang et al.[89] have found that SSTF attenuated EAE disease severity, accompanied by enhanced Treg frequency and level of Treg-associated cytokines (IL-10 and TGF-β), as well as downregulated Th17 frequency and expression of Th17-related cytokines (IL-17 and IL-23).

    It is reported that the essential oils from the aerial parts of S. baicalensis showed toxicity against booklice (Liposcelis bostrychophila) with an LC50 of 141.37 μg/cm2. The components of myristate, caryophyllene, eugenol, and caryophyllene oxide displayed dramatic toxicity against the L. bostrychophila, with LC50 values of 290.34, 104.32, 85.75, and 21.13 μg/cm2, respectively[31].

    Guo & Xu[90] have found that SSTF could inhibit the proliferation of Hela cells obviously (p< 0.01). Tang et al.[10] have found that SSTF could play an anti-colon cancer role by up-regulated the expressions of Cleaved Caspase-3 and the ratio of Bax/Bcl-2 (p < 0.05 and 0.01) and significantly down-regulated the expressions of MMP-2 and MMP-9 in HCT116 cells (p < 0.05 and 0.01). Recently, Shen et al.[6] studied the chemopreventive effects of HQT against AOM-induced preneoplastic colonic aberrant crypt foci in rats and found HQT inhibits AOM-induced aberrant crypt foci formation by modulating the gut microbiota composition, inhibiting inflammation and improving metabolomic disorders.

    Cardiovascular disease is one of the most important threats to human health. Hyperlipidemia is a major risk factor for atherosclerosis, which can cause various cardiovascular and cerebrovascular diseases. Different doses of SSTF (50, 100, and 200 mg/kg) can effectively reduce the body weight increase of rats with hypertriglyceridemia, reduce the serum levels of triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), which indicate that SSTF has the effect of regulating blood lipid[91].

    Oxidative stress is important in developing tissue damage in several human diseases[92]. The antioxidant capacities of separated organs (flower, leaf, stem, and root) of S. baicalensis were conducted by DPPH, ABTS+, and RP methods, respectively. The results showed that the antioxidant activity of the root (66.9 ± 0.3, 121.6 ± 0.5, and 80.2 ± 0.4 μg/mL) was the highest, followed by the leaf (68.4 ± 1.3, 128.2 ± 2.1, and 135.8 ± 2.0 μg/mL), stem (127.8 ± 3.1, 199.2 ± 1.7, and 208.2 ± 8.3 μg/mL) and flower (129.8 ± 6.3, 285.7 ± 4.7, and 380.3 ± 14.2 μg/mL)[93]. The antioxidant activities of the extracts of the aerial part of S. baicalensis and separated organs were conducted by DPPH assay and reducing power method with the antioxidant ability 7.73–8.83 mg TE/g DW and 51.48–306.09 mg TE/g DW, respectively. The content of total polyphenols and total flavonoids were significantly positively correlated with the reducing power[94]. Liu et al.[95] found that the flavonoids extracted from the stems and leaves of S. baicalensis (SSF, 18.98, 37.36, and 75.92 μg/mL) could protect rat cortical neurons against H2O2-induced oxidative injury in a dose-dependent manner. Cao et al. found that SSTF could alleviate the damage of human umbilical vascular endothelial cells injured by H2O2 and reduce their apoptosis, which may be related to the increasing level of Bcl-2[96].

    Preventive treatment with SSTF (50, 100, and 200 mg/kg) could significantly inhibit the blood glucose increase induced by alloxan in mice, and SSTF treatment could reduce the blood glucose level in diabetic mice. Both the prevention group and the treatment group could increase the activity of serum superoxide dismutase and decrease the content of malondialdehyde[97]. Liu et al.[98] found that SSTF (75 and 150 mg/kg) could significantly reduce blood glucose and blood lipid and improve insulin resistance in type 2 diabetic rats with hyperlipidemia.

    Yang et al.[99] found that SSTF (35 mg/kg/d, 8 weeks) could resist hepatic fibrosis by inhibiting the expression of α-smooth muscle actin in Hepatic Stellate Cells. In vivo, it is reported that SSTF (50, 100, and 200 mg/kg) could significantly reduce alanine transaminase activities in serum, increase the expression of superoxide dismutase and reduce the content of malondialdehyde in acute hepatic injury mice induced by carbon tetrachloride and ethanol[100].

    These findings suggest that S. baicalensis stem and leaf extract hold promise in promoting various aspects of health, including immune modulation, antioxidant activity, anti-tumor effects, cardiovascular health, oxidative stress protection, and more. However, further research, including clinical studies, is necessary to better understand the full therapeutic potential and safety of these effects in human applications.

    Flavonoids are considered the main active components in HQT. As the active substance basis of HQT, the safety of SSTF has also been investigated. After 90 d of oral administration of SSTF (0.5, 1, and 2 g/kg) to rats, no abnormal changes were observed in all indexes, and no delayed toxic reactions or obvious toxic reactions were observed, indicating that the toxicity of SSTF is low[101]. The LD50 value of SSTF was 14.87 g/kg is equivalent to 68.5 times the maximum dose in the pharmacodynamic test of mice, and the experiment confirms the safety of oral administration of the SSTF. Intraperitoneal injection of SST showed certain toxicity in mice, with an LD50 value of 732.11 mg/kg[102]. Liu et al.[103] conducted a systematic safety assessment experiment on the aqueous extract of S. baicalensis stem and leaves based on the China National Standard 'Guidelines for the Safety Evaluation of Food Toxicology (GB15193-2014)'. The results indicated that S. baicalensis stem and leaves are non-toxic, non-teratogenic, and non-mutagenic. Acute toxicity tests in mice revealed a Maximum Tolerated Dose of 15.0 g/kg. A 90-d feeding trial showed no changes in toxicological damage in animals, even at a high dosage of 8.333 g/kg (equivalent to 100 times the recommended human daily intake), suggesting the safety and non-toxicity of consuming S. baicalensis stem and leaves. In addition, HQT has long been used in folklore, and no toxicity has been reported.

    These findings collectively indicate that HQT is generally safe for consumption. However, as with any herbal product, it's important to follow recommended dosages and consult healthcare professionals, especially for individuals with pre-existing health conditions or medications.

    In recent years, HQT, a non-Camellia tea with a long history in China, has attracted attention due to its diverse pharmacological activities. Among various Scutellaria species, S. baicalensis is the most extensively studied and cultivated source for HQT. The aerial parts, including flowers, stems, and leaves, serve as the principal source of HQT preparation. HQT are rich in flavonoids and volatile components with various beneficial effects. To date, about 295 compounds have been identified from HQT, including approximately 54 flavonoid compounds and 145 volatile components identified online. The current research on the activity of HQT primarily focuses on flavonoid compounds, with limited studies on the larger quantity of volatile oil compounds.

    Additionally, the processing and brewing techniques used to prepare HQT may influence the bioactivity of its flavonoid content, although few studies have investigated this. More research is necessary to optimize the processing and brewing techniques to maximize the health benefits of HQT. Comparative studies reveal that the aerial parts of S. baicalensis, while sharing similarities with the roots, contain varying flavonoid compositions. Limited research on S. scordifolia, S. amoena, and S. viscidula, indicates the presence of comparable flavonoid compounds in their aerial parts. Although individual flavonoids like baicalin, wogonin, and scutellarin have demonstrated various therapeutic properties, it is essential to consider the synergistic effects of these compounds when consumed together in the form of tea. These findings contribute to laying the groundwork for quality assessment of HQT and offer insights into potential health benefits.

    HQT is mainly derived from the aerial parts of S. baicalensis. Recent studies have increasingly recognized the pharmacological value of the aerial parts of S. baicalensis. Preliminary pharmacological studies have shown that the aerial parts of S. baicalensis may possess beneficial activities in antioxidant, anti-tumor, antiviral, anti-bacterial, protection of ischemia-reperfusion injured neural function, neuroprotective effects against brain injury, and blood lipid regulation. These findings suggest that the value of using HQT may be attributed to these pharmacological activities. Although HQT is generally safe for consumption, further investigation is required to understand its safety profile, particularly in special populations such as pregnant or lactating women, children, and individuals with pre-existing medical conditions. Additionally, potential interactions between the flavonoids in HQT and conventional medications must be examined to ensure their safe and effective use with pharmaceutical treatments. In addition, although the safe dose of HQT on rodents has been studied, the safe dose for humans has yet to be determined.

    In conclusion, these initial research results support the potential health benefits of HQT and encourage more in-depth studies on its raw materials. Further studies are necessary to elucidate the synergistic effects of the flavonoids in HQT, optimize the processing and brewing techniques for maximum bioactivity, and investigate the safety profile and potential interactions with conventional medications. A comprehensive understanding of HQT will contribute to developing evidence-based recommendations for promoting health and well-being.

    The authors confirm contribution to the paper as follows: Conceptualization and writing: Quan Y, Li Z, Meng X, Li P, Shen J; Figure and table modification: Quan Y, Li Z, Meng X, Li P; review and editing: Wang Y, He C, Shen J. All authors reviewed the results and approved the final version of the manuscript.

    The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    This research was funded by the Shandong Provincial Natural Science Foundation, China (ZR2022QH147, ZR2022QH165) and the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (No. ZYYCXTD-D-202005).

  • The authors declare that they have no conflict of interest.

  • Supplemental Table S1 Primer sequences used in RT-qPCR.
    Supplemental Table S2 The nomenclature and physicochemical properties of MsCYP450 genes.
    Supplemental Fig. S1 Synteny analysis of CYP450 genes form alfalfa with rice, Arabidopsis, soybean, and thistle alfalfa.
    Supplemental Fig. S2 Ka and Ks analysis of plant CYP450 duplicated genes. The number (A), Ka (B), Ks (C), and Ka/Ks (D) of CYP450 duplicated genes. Segment, segmental duplicated gene pairs; tandem, tandem duplicated gene pairs; Ms-Os, CYP450 duplicated gene pairs between alfalfa and rice; Ms-At, CYP450 duplicated gene pairs between alfalfa and Arabidopsis; Ms-Gm, CYP450 duplicated gene pairs between alfalfa and soybean; Ms-Mt, duplicated gene pairs between alfalfa and thistle alfalfa.
    Supplemental Fig. S3 Different cis-elements in the promoters of nine MsCYP genes. White squares represent the absence of a cis-element in the promoter of MsCYP genes, and the number in the square represents the number of cis-elements in the promoter.
  • [1]

    Ashraf M, Wu L. 1994. Breeding for salinity tolerance in plants. Critical Reviews in Plant Sciences 13:17−42

    doi: 10.1080/07352689409701906

    CrossRef   Google Scholar

    [2]

    Croser C, Renault S, Franklin J, Zwiazek J. 2001. The effect of salinity on the emergence and seedling growth of Picea mariana, Picea glauca, and Pinus banksiana. Environmental Pollution 115:9−16

    doi: 10.1016/S0269-7491(01)00097-5

    CrossRef   Google Scholar

    [3]

    Tang X, Mu X, Shao H, Wang H, Brestic M. 2015. Global plant-responding mechanisms to salt stress: physiological and molecular levels and implications in biotechnology. Critical Reviews in Biotechnology 35:425−37

    doi: 10.3109/07388551.2014.889080

    CrossRef   Google Scholar

    [4]

    Munns R, Gilliham M. 2015. Salinity tolerance of crops – what is the cost? New Phytologist 208:668−73

    doi: 10.1111/nph.13519

    CrossRef   Google Scholar

    [5]

    Acosta-Motos JR, Diaz-Vivancos P, Álvarez S, Fernández-García N, Sanchez-Blanco MJ, et al. 2015. Physiological and biochemical mechanisms of the ornamental Eugenia myrtifolia L. plants for coping with NaCl stress and recovery. Planta 242:829−46

    doi: 10.1007/s00425-015-2315-3

    CrossRef   Google Scholar

    [6]

    Li X, Wei Y, Moore KJ, Michaud R, Viands DR, et al. 2011. Association mapping of biomass yield and stem composition in a tetraploid alfalfa breeding population. The Plant Genome 4:24−35

    doi: 10.3835/plantgenome2010.09.0022

    CrossRef   Google Scholar

    [7]

    Luo D, Zhou Q, Wu Y, Chai X, Liu W, et al. 2019. Full-length transcript sequencing and comparative transcriptomic analysis to evaluate the contribution of osmotic and ionic stress components towards salinity tolerance in the roots of cultivated alfalfa (Medicago sativa L.). BMC Plant Biology 19:32

    doi: 10.1186/s12870-019-1630-4

    CrossRef   Google Scholar

    [8]

    Ma Q, Xu X, Wang W, Zhao L, Ma D, et al. 2021. Comparative analysis of alfalfa (Medicago sativa L.) seedling transcriptomes reveals genotype-specific drought tolerance mechanisms. Plant Physiology Biochemistry 166:203−14

    doi: 10.1016/j.plaphy.2021.05.008

    CrossRef   Google Scholar

    [9]

    Sun W, Ma Z, Liu M. 2020. Cytochrome P450 family: genome-wide identification provides insights into the rutin synthesis pathway in Tartary buckwheat and the improvement of agricultural product quality. International Journal of Biological Macromolecules 164:4032−45

    doi: 10.1016/j.ijbiomac.2020.09.008

    CrossRef   Google Scholar

    [10]

    Nelson D, Werck-Reichhart D. 2011. A P450-centric view of plant evolution. The Plant Journal 66:194−211

    doi: 10.1111/j.1365-313X.2011.04529.x

    CrossRef   Google Scholar

    [11]

    Khanom S, Jang J, Lee OR. 2019. Overexpression of ginseng cytochrome P450 CYP736A12 alters plant growth and confers phenylurea herbicide tolerance in Arabidopsis. Journal of Ginseng Research 43:645−53

    doi: 10.1016/j.jgr.2019.04.005

    CrossRef   Google Scholar

    [12]

    Bak S, Paquette SM, Morant M, Morant AV, Saito S, et al. 2006. Cyanogenic glycosides: a case study for evolution and application of cytochromes P450. Phytochemistry Reviews 5:309−29

    doi: 10.1007/s11101-006-9033-1

    CrossRef   Google Scholar

    [13]

    Podust LM, Sherman DH. 2012. Diversity of P450 enzymes in the biosynthesis of natural products. Natural Product Reports 29:1251−66

    doi: 10.1039/c2np20020a

    CrossRef   Google Scholar

    [14]

    Ghosh S. 2017. Triterpene structural diversification by plant cytochrome P450 enzymes. Frontiers in Plant Science 8:1886

    doi: 10.3389/fpls.2017.01886

    CrossRef   Google Scholar

    [15]

    Banerjee A, Hamberger B. 2018. P450s controlling metabolic bifurcations in plant terpene specialized metabolism. Phytochemistry Reviews 17:81−111

    doi: 10.1007/s11101-017-9530-4

    CrossRef   Google Scholar

    [16]

    Hansen CC, Nelson DR, Møller BL, Werck-Reichhart D. 2021. Plant cytochrome P450 plasticity and evolution. Molecular Plant 14:1244−65

    doi: 10.1016/j.molp.2021.06.028

    CrossRef   Google Scholar

    [17]

    Shen C, Du H, Chen Z, Lu H, Zhu F, et al. 2020. The chromosome-level genome sequence of the autotetraploid alfalfa and resequencing of core germplasms provide genomic resources for alfalfa research. Molecular Plant 13:1250−61

    doi: 10.1016/j.molp.2020.07.003

    CrossRef   Google Scholar

    [18]

    Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, et al. 2020. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Molecular Plant 13:1194−202

    doi: 10.1016/j.molp.2020.06.009

    CrossRef   Google Scholar

    [19]

    Zhang X, Henriques R, Lin SS, Niu QW, Chua NH. 2006. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocals 1:641−46

    doi: 10.1038/nprot.2006.97

    CrossRef   Google Scholar

    [20]

    Mainali HR, Chapman P, Dhaubhadel S. 2014. Genome-wide analysis of Cyclophilin gene family in soybean (Glycine max). BMC Plant Biology 14:282

    doi: 10.1186/s12870-014-0282-7

    CrossRef   Google Scholar

    [21]

    Xiong R, He T, Wang Y, Liu S, Gao Y, et al. 2021. Genome and transcriptome analysis to understand the role diversification of cytochrome P450 gene under excess nitrogen treatment. BMC Plant Biology 21:447

    doi: 10.1186/s12870-021-03224-x

    CrossRef   Google Scholar

    [22]

    Wei K, Chen H. 2018. Global identification, structural analysis and expression characterization of cytochrome P450 monooxygenase superfamily in rice. BMC Genomics 19:35

    doi: 10.1186/s12864-017-4425-8

    CrossRef   Google Scholar

    [23]

    Levsh O, Pluskal T, Carballo V, Mitchell AJ, Weng JK. 2019. Independent evolution of rosmarinic acid biosynthesis in two sister families under the Lamiids clade of flowering plants. Journal of Biological Chemistry 294:15193−205

    doi: 10.1074/jbc.RA119.010454

    CrossRef   Google Scholar

    [24]

    Mao G, Seebeck T, Schrenker D, Yu O. 2013. CYP709B3, a cytochrome P450 monooxygenase gene involved in salt tolerance in Arabidopsis thaliana. BMC Plant Biology 13:169

    doi: 10.1186/1471-2229-13-169

    CrossRef   Google Scholar

    [25]

    Balusamy SR, Rahimi S, Yang DC. 2019. Characterization of squalene-induced PgCYP736B involved in salt tolerance by modulating key genes of abscisic acid biosynthesis. International Journal of Biological Macromolecules 121:796−805

    doi: 10.1016/j.ijbiomac.2018.10.058

    CrossRef   Google Scholar

    [26]

    Krishnamurthy P, Vishal B, Ho WJ, Lok FCJ, Lee FSM, et al. 2020. Regulation of a cytochrome P450 gene CYP94B1 by WRKY33 transcription factor controls apoplastic barrier formation in roots to confer salt tolerance. Plant Physiology 184:2199−215

    doi: 10.1104/pp.20.01054

    CrossRef   Google Scholar

    [27]

    Wang L, Wen S, Wang R, Wang C, Gao B, et al. 2021. PagWOX11/12a activates PagCYP736A12 gene that facilitates salt tolerance in poplar. Plant Biotechnology Journal 19:2249−60

    doi: 10.1111/pbi.13653

    CrossRef   Google Scholar

    [28]

    Wang C, Yang Y, Wang H, Ran X, Li B, et al. 2016. Ectopic expression of a cytochrome P450 monooxygenase gene PtCYP714A3 from Populus trichocarpa reduces shoot growth and improves tolerance to salt stress in transgenic rice. Plant Biotechnology Journal 14:1838−51

    doi: 10.1111/pbi.12544

    CrossRef   Google Scholar

    [29]

    Kim SK, You YN, Park JC, Joung Y, Kim BG, et al. 2012. The rice thylakoid lumenal cyclophilin OsCYP20-2 confers enhanced environmental stress tolerance in tobacco and Arabidopsis. Plant Cell Reports 31:417−26

    doi: 10.1007/s00299-011-1176-x

    CrossRef   Google Scholar

    [30]

    Kumari S, Joshi R, Singh K, Roy S, Tripathi AK, et al. 2015. Expression of a cyclophilin OsCyp2-P isolated from a salt-tolerant landrace of rice in tobacco alleviates stress via ion homeostasis and limiting ROS accumulation. Functional & Integrative Genomics 15:395−412

    doi: 10.1007/s10142-014-0429-5

    CrossRef   Google Scholar

    [31]

    Zhu C, Wang Y, Li Y, Bhatti KH, Tian Y, et al. 2011. Overexpression of a cotton cyclophilin gene (GhCyp1) in transgenic tobacco plants confers dual tolerance to salt stress and Pseudomonas syringae pv. tabaci infection. Plant Physiology Biochemistry 49:1264−71

    doi: 10.1016/j.plaphy.2011.09.001

    CrossRef   Google Scholar

    [32]

    Sekhar K, Priyanka B, Reddy VD, Rao KV. 2010. Isolation and characterization of a pigeonpea cyclophilin (CcCYP) gene, and its over-expression in Arabidopsis confers multiple abiotic stress tolerance. Plant, Cell & Environment 33:1324−38

    doi: 10.1111/j.1365-3040.2010.02151.x

    CrossRef   Google Scholar

  • Cite this article

    Zhang X, Xue L, Chen R, Ma Q, Ma D, et al. 2023. Genome-wide identification of the cytochrome P450 family and analysis of CYP regarding salt tolerance in Medicago sativa L. Grass Research 3:21 doi: 10.48130/GR-2023-0021
    Zhang X, Xue L, Chen R, Ma Q, Ma D, et al. 2023. Genome-wide identification of the cytochrome P450 family and analysis of CYP regarding salt tolerance in Medicago sativa L. Grass Research 3:21 doi: 10.48130/GR-2023-0021

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Genome-wide identification of the cytochrome P450 family and analysis of CYP regarding salt tolerance in Medicago sativa L.

Grass Research  3 Article number: 21  (2023)  |  Cite this article

Abstract: With the ever-changing environment and climate, high salinity has become a major environmental issue that threatens crop quality and yield. Alfalfa (Medicago sativa L.) is a perennial forage crop planted worldwide that has a well-developed root system and salt tolerance. Cytochrome P450 monooxygenase (CYP450) genes play important roles in flavonoid synthesis, plant growth, and development. However, few studies have focused on CYP450s in forage grass, especially the role of CYP450 genes in plant resistance to environmental stresses, such as drought and high salinity. In this study, 376 menbers in MsCYP family genes were identified using hmmsearch and BLASTP in the alfalfa protein database using the AtCYP450 protein sequence. Then by exploring MsCYP gene structures, tandem and segmental duplication events, and evolutionary relationships with CYP450s in other plants, potential MsCYPs that respond to salt stress were screened. Candidate genes were selected for transient expression in tobacco and heterologous overexpression in Arabidopsis for salinity response. This study provides a foundation for verifying the function of MsCYPs in improving the quality of agricultural products.

    • 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[68]. 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.

    • The genome sequence was downloaded from the Alfalfa Genome Project website (https://Figureshare.com/articles/dataset/Medicago_sativa_genome)[17]. The HMM (Hidden Markov Model) of the CYP450 domain (PF00067) was obtained from Pfam (http://pfam.xfam.org/family/PF00067#curationBlock) and used to search for homologous sequences in the alfalfa protein sequence file using hmmer-3.0-windows. Furthermore, the CYP450 sequences of Arabidopsis were used to blast the alfalfa protein file (e-value 1e−5). The total protein sequences were obtained by combining these two methods. To confirm the reliability of the search, all candidate sequences obtained were checked using Pfam (http://pfam.xfam.org/search#tabview=tab1) and NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) for conserved domain identification. Redundant sequences were removed, and nonredundant CYP450 sequences were used for sequence alignment and further analyses, including the determination of chromosomal location, isoelectric point, and subcellular localization.

    • CYP450 protein sequences were extracted from Arabidopsis thaliana and alfalfa. The sequences of CYP450 proteins from all plant species were compared using the MAFFT software. The FastTree software was used to construct a phylogenetic tree using the largest natural control with a bootstrap value of 1,000. The gene and coding sequences of MsCYP genes were used to analyze the gene structure using TBtools[18].

    • Duplicate genes in the MsCYP gene family were identified using one-step MCScanX in TBtools v1.098746. Simple Ka/Ks calculators (NG) were employed to calculate Ka and Ks using TBtools v1.098746. The Arabidopsis genome sequence was downloaded from https://www.ncbi.nlm.nih.gov/genome/?term=Arabidopsis+thaliana; the rice genome sequence was downloaded from https://data.jgi.doe.gov/refine-download/phytozome?organism=Osativa&expanded=Phytozome-323; the Glycine max genome sequence was downloaded from https://ngdc.cncb.ac.cn/search/?dbId=gwh&q=GWHAAEV00000000&page=1; and the M. truncatula genome sequence was downloaded from https://www.ncbi.nlm.nih.gov/genome/?term=Medicago+truncatula+. Pairs of duplicated genes in M. sativa, Arabidopsis, Oryza sativa, G. max, and M. truncatula were analyzed in the same way as described above. The relationships between duplicates in the MsCYP gene family were plotted using Advanced Circo in TBtools v1.098746, and the relationships between duplicates among these species were plotted using multiple synteny plots in TBtools v1.098746.

    • Promoter sequences (2,000 bp upstream of the MsCYP genes) were obtained from the alfalfa genome using Gtf/Gff3 Sequence Extract and Fasta Extract in TBtools v1.098746. The upstream 1,500 bp sequences were prefetched as promoters and submitted to PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to identify cis-acting elements.

    • M. sativa (Zhong Mu No. 1) seeds were sown in soil and grown for 10 d, then hydroponically cultivated for 12 d. Tobacco (Nicotiana benthamiana), and A. thaliana were cultivated using nutrient soil in the Plant Growth Laboratory of Ningxia University (China). The culture temperature was 25 °C, and the photoperiod was 16 h light and 8 h dark. The roots of 22-day-old alfalfa seedlings were exposed to 200 mM NaCl for 2 h and recovered using hydroponic experiments. The samples were collected at three time points — before salt treatment (N0), after salt treatment for 2 h (N2), and after rehydration for 3 d (NH) — from three different plants and immediately stored at −80 °C until further use.

    • RNA was extracted from frozen samples using a MiniBEST Plant RNA Extraction Kit (Takara Bio Inc., Shiga, Japan). cDNA was generated with PrimeScript™ RT Master Mix (Takara Bio Inc.), and RT-qPCR was performed in triplicate using a Roche Light Cycler 480 (Roche, Basel, Switzerland) with TB Green® Premix Ex Taq™ II (Tli RNaseH Plus; Takara Bio Inc.) according to the manufacturer’s instructions. Gene-specific primers (Supplemental Table S1) were designed to amplify the nine MsCYP genes using Primer Premier5.0. Actin was used as an internal control gene, and the relative expression levels of these MsCYPs were estimated via the 2−ΔΔCᴛ method.

    • A library was sequenced on an Illumina Novaseg 6000 platform. Fastp was used to remove adapter and low quality reads from raw data. The clean reads were then mapped to the alfalfa genome using Hisat2. Reads counts was obtained with Htseq-count, and differential expression analysis was performed using the DESeq R package. Reads counts were transformed into FPKM with R.

    • The full-length coding sequence of MsCYP273 was amplified from the cDNA of alfalfa, and was inserted upstream of GFP harbored by the pGreen0029 vector via homologous recombination. Relevant negative and positive controls were produced simultaneously. The resulting constructs were then transiently transformed into tobacco leaves. Fluorescence was detected using an SP8 confocal microscope (Leica) at excitation wavelengths of 488 nm, and emission wavelengths of 495–545 nm for GFP, 590–670 nm for chloroplasts. To generate MsCYP273 overexpression lines, the coding sequence of the MsCYP273 protein was inserted into the pCAMBIA1300 plasmid, which was stably transformed into Arabidopsis using A. tumefaciens (GV3101) via the Arabidopsis floral dip method, as previously described[19].

    • To identify CYP450 genes in the alfalfa genome, an HMM search was performed against the alfalfa protein database using BLASTP. A total of 376 MsCYP genes were identified and named MsCYP1–MsCYP376 according to their chromosomal locations. The number of MsCYP amino acid sequences ranged from 120–984. The molecular weight of MsCYP proteins ranged from 13.62–110.08 kDa, and the isoelectric point ranged from 4.53–10.17. The WoLF PSORT tool predicted that approximately 192 (51.06%) and 100 (26.60%) MsCYPs were located in the chloroplast and cytosol, respectively; the remaining MsCYPs were located in the nucleus, mitochondria, and peroxisomes (Supplemental Table S2).

    • To investigate the evolutionary relationships of the CYP gene family in alfalfa, the CYP450 sequences of A. thaliana, M. truncatula, and M. sativa were used to construct a phylogenetic tree using FastTree (Fig. 1). The MsCYP proteins are divided into nine major clans: CYP71, CYP51, CYP72, CYP74, CYP85, CYP86, CYP97, CYP710, and CYP711. The CYP71 clan was of the A-type, while the other clans were of the non-A-type. CYP71, CYP86, CYP85, CYP72, CYP97, and CYP74 contained 236, 48, 35, 39, three, and two MsCYP proteins, respectively. The CYP51, CYP710, and CYP711 clans contained only one MsCYP protein (Fig. 1). Many clans, including CYP71, CYP86, CYP72, and CYP74, were significantly more abundant in M. sativa than A. thaliana or M. truncatula. In the CYP711, CYP97, and CYP51 clans, the gene phylogeny roughly followed the species phylogeny, with the genes of M. sativa displaying a sister-group relationship with M. truncatula, and one CYP450 gene in Arabidopsis closely related to alfalfa homologs. In particular, the phylogeny of the CYP85 clan was more complex, suggesting multiple duplication events during the evolution of the dicot CYP450 gene family.

      Figure 1. 

      Phylogenetic analysis of CYP genes from alfalfa, thistle alfalfa, and Arabidopsis. Different colors represent different groups.

    • To understand their functional regions, conserved motifs of the MsCYPs were analyzed. The motif distribution of each MsCYP protein was analyzed using MEME, and most members of the MsCYP gene family contained motifs 5, 3, 7, 10, and 11. Additionally, the arrangement and composition of the motifs were consistent with the phylogenetic tree results. For example, most CYP71 clan members contain motifs 8, 9, 2, 5, 3, 7, 4, 10, 1, and 6; however, these motifs were not identified in CYP76, CYP200, CYP208, or CYP259. All MsCYP proteins contain conserved CYP450 or P450 superfamily domains, and each MsCYP coding sequence is separated by introns ranging from 0–16. Most MsCYP proteins in each sample contained similar numbers of introns; most members of the CYP97, CYP72, and CYP85 clans have more introns, whereas members of the other clans have fewer introns (Fig. 2). Generally, the motif composition of the members within each clan was similar, indicating that the protein structure was highly conserved and further validating the reliability of the phylogenetic tree (Fig. 2).

      Figure 2. 

      Phylogeny, conserved motifs, and exon–intron structure of MsCYP genes.

      All MsCYP proteins contained either the P450 superfamily or CYP450 domain, indicating that they had a similar nature or function. The MsCYP sequence contained introns 1–13, and both the number and phase of MsCYP genes were well conserved in the same clan. For example, each MsCYP gene in CYP706A contained two phase 0 introns. The number of introns in the MsCYP gene of CYP724A ranged from 9–10 (Fig. 2).

    • Based on newly published genome-wide data from alfalfa, the distribution of MsCYPs on chromosomes was analyzed. The 376 MsCYP genes were not evenly distributed on the eight chromosomes, with more genes identified on chromosomes 1 and 8 (59 and 58 genes, respectively). A total of 56 MsCYP genes were located on chromosomes 3 and 4, 44 on chromosome 5, 31 on chromosomes 2 and 6, and 33 on chromosome 7 (Fig. 3).

      Figure 3. 

      Chromosomal locations of MsCYP genes. Tandem duplicates are marked with curved lines.

      To explore the relationship between the evolutionary expansion of the MsCYP gene family and whole-genome duplication events in alfalfa, tandem duplication and segmental replication events in MsCYP genes were analyzed using the one-step MCScanX method. A total of 112 MsCYP genes in the MsCYP family were involved in tandem duplication, accounting for 29.8% of all MsCYP genes. A total of 64 tandem duplication pairs were obtained, which were distributed on eight chromosomes; most occurred on chromosomes 3 and 4 (Fig. 3). Only 37 genes (approximately 10%) were involved in segment duplication, and 19 pairs of segment duplication genes were obtained; these were distributed on chromosomes 1, 3, 4, 5, 6, 7, and 8. Chromosome 2 was not involved in segment duplication (Fig. 4). Tandem duplication events are thought to be a major driver of the expansion of the MsCYP family. Notably, six genes (CYP30, CYP106, CYP121, CYP165, CYP191, and CYP203) were involved in both segment and tandem duplications.

      Figure 4. 

      Synteny analysis of alfalfa CYP genes. Different segmental duplicate pairs are linked with different colors.

      To further explore the evolutionary processes of the MsCYP family, four comparative syntenies of alfalfa with rice, Arabidopsis, soybean, and thistle alfalfa were constructed. Alfalfa and thistle alfalfa shared the most orthologous pairs with up to 173 pairs of orthologous CYP450s, followed by alfalfa and soybean with 163 pairs. We also identified 44 pairs of orthologous CYP450s between alfalfa and Arabidopsis, and 15 pairs between alfalfa and rice. A closer relationship has been suggested between alfalfa and thistle alfalfa/soybean than between Arabidopsis and rice (Supplemental Fig. S1).

      To explore the selection pressures acting on the CYP450 gene family, Ka, Ks, and Ka/Ks values were calculated for these gene pairs. All Ka/Ks values of the duplicated gene pairs from the alfalfa CYP450 gene family were < 0.8, excluding MsCYP2/MsCYP256. Some orthologous gene pairs had Ka/Ks values < 0.7; however, the remaining orthologous gene pairs were highly divergent between alfalfa and Arabidopsis/soybean/thistle alfalfa. All orthologous gene pairs were highly divergent between alfalfa and rice (Supplemental Fig. S2). These results suggest that the MsCYP gene family underwent strong purifying selection.

    • Using RNA-seq and RT-qPCR analyses before and after salt stress and rehydration (N0, N2, and NH, respectively), we identified nine MsCYP genes (MsCYP273, MsCYP272, MsCYP275, MsCYP266, MsCYP271, MsCYP267, MsCYP268, MsCYP89, and MsCYP274) believed to play vital roles in the response to salt stress. These genes were highly expressed in the N2 group and exhibited reduced expression in the N0 and NH groups (Fig. 5).

      Figure 5. 

      Expression pattern of nine key MsCYP genes. The column indicates RNA-seq data, the red line indicates RT-qPCR data, and the error bar indicates the standard error of the mean (three biological replicates and three technical replicates). **, p < 0.01. ns, not significant. N0 refers to gene expression before salt treatment; N2 refers to gene expression after salt treatment for 2 h; and NH refers to gene expression after rehydration for 3 d.

      To further predict the function of these nine MsCYP genes, cis-elements in the promoters were analyzed, and 72 elements were identified (Supplemental Fig. S3). Most elements were associated with development, especially the light response, including the 3-AF1 binding site, AAAC motif, ACE, and AE box. CAAT and TATA boxes were found in the promoter regions of all MsCYP genes. The MYB-binding site, known for its role in the drought response, was identified in MsCYP273, MsCYP272, MsCYP275, MsCYP266, MsCYP268, and MsCYP89. Among these, MsCYP273 was selected for transient expression in tobacco and heterologous overexpression in Arabidopsis. MsCYP273 was mainly located in the chloroplasts, with some distribution in the cell membrane and cytoplasm (Fig. 6b). In addition, five MsCYP273 overexpression lines were obtained, all of which had an expression of MsCYP273 (Fig. 6a). Compared to the wild type, MsCYP273 overexpression lines demonstrated stronger resistance to 75 mM NaCl stress (Fig. 6c, d).

      Figure 6. 

      Analysis of the MsCYP273 protein. (a) RT-PCR results of five MsCYP273 overexpressing lines. (b) Subcellular localization of MsCYP273 protein. Bar = 20 μm. Bright: Bright field; GFP: green fluorescent protein of 495-545 nm; CHF: chloroplast autofluorescence of 590–670 nm. (c) Phenotype of MsCYP273 overexpressing lines (MsCYP273-line2 and MsCYP273-line10) under salt stress. (d) Root length analysis of MsCYP273 overexpressing lines (MsCYP273-line2 and MsCYP273-line10) under salt stress. Bar = 1 cm. ***, p < 0.001.

    • Cyclophilins are involved in several physiological processes, including protein transport, transcriptional regulation, signal transduction, mRNA splicing, cell apoptosis, and stress response. The total number of CYP450 genes in a single plant species is usually higher than in animals[16]. Comprehensive analyses of CYP450 gene families in several plants have been published, and gene duplication has played an important role in the expansion of the gene family. A total of 62 CYP450 genes were identified in soybeans (G. max); 54 were clustered in pairs (27 pairs) in the phylogenetic tree[20]. In the apple (Malus domestica) genome, 30 CYP450 genes were identified; there were no tandem duplicated gene pairs, and 10 segmental duplicated gene pairs. There are 188 CYP450 genes in Panax notoginseng, with eight tandem and 11 segmental duplication events[21]. In the Tartary buckwheat (Fagopyrum tataricum) genome, 285 CYP450 genes have been identified, with 62 pairs of tandemly duplicated and 18 pairs of segmentally duplicated genes. A total of 355 CYP450 genes were identified in rice (O. sativa); more than half of these genes were found in 53 tandem duplicated gene clusters, while 55 OsCYP450s were distributed in segmental duplication blocks[22]. In this study, 376 MsCYP genes were identified in alfalfa; 144 genes were involved in gene duplication, resulting in 64 tandemly duplicated gene clusters and 19 segmentally duplicated gene pairs (Figs 3 & 4). Thus, gene duplication has played a significant role in the evolution of the plant CYP450 gene family. In particular, the Tartary buckwheat, alfalfa, and rice CYP450 gene families are prone to differential expansion due to tandem duplication during evolution.

    • Gene duplication is the main source of redundancy and functional specialization in the evolution of plant genomes. For example, the duplication and neofunctionalization of CYP98 subfamily genes have enabled hydroxylation during rosmarinic acid biosynthesis in some lamiid plants[23]. The CYP450 KLUH/CYP78A5 and its homolog, CYP78A7, promote organ growth via a noncell-autonomous signaling pathway. TaCYP81D5, a tandemly arranged CYP81D gene, confers salinity tolerance by scavenging reactive oxygen species (ROS) in bread wheat. In the present study, we identified nine key MsCYP genes (MsCYP273, MsCYP272, MsCYP275, MsCYP266, MsCYP271, MsCYP267, MsCYP268, MsCYP89, and MsCYP274), all of which were clustered in the CYP71 clan and may play vital roles in the response to salt stress (Fig. 1). Furthermore, MsCYP266, MsCYP273, and MsCYP275 clustered closely in the CYP736A subfamily, whereas MsCYP268, MsCYP272, MsCYP274, MsCYP271, and MsCYP267 clustered closely in the CYP71D subfamily. In particular, MsCYP266/MsCYP273 and MsCYP267/MsCYP274 were segmentally duplicated gene pairs, both of which showed similar expression patterns under salt stress conditions. Thus, further studies are needed to explore the functional relevance of similar duplicated genes to confirm whether neofunctionalization and subfunctionalization of alfalfa CYP450 genes influence the adaptation of alfalfa to diverse conditions.

    • Salt stress is a major environmental factor that can adversely alter plant growth and development and reduce global crop production. CYP450 enzymes play significant roles in the salt stress response of plants. In this study, we identified nine MsCYP genes that exhibited increased expression during salt stress, and decreased expression during recovery (Fig. 5); this was consistent with the results of previous studies. For example, AtCYP709B3 is markedly induced by salt stress and remains highly expressed, while mutant Arabidopsis plants are sensitive to ABA and salt during germination[24]. The overexpression of PgCYP736B in Arabidopsis confers enhanced resistance to salt stress by decreasing hydrogen peroxide accumulation, thereby increasing carotenoid levels and abscisic acid biosynthesis gene expression[25]. The heterologous expression of AoCYP94B1 in Arabidopsis and rice seedlings confers salt tolerance by enhancing root suberin deposition[26]. PagWOX11/12a induces the expression of PagCYP736A12 to modulate ROS scavenging, thus enhancing salt tolerance in 84 K poplar (Populus alba × P. glandulosa)[27]. PtCYP714A3 is markedly induced by salt stress, and transgenic rice plants exhibit reduced shoot growth and enhanced salt tolerance[28]. OsCyP20-2, located in the thylakoid, is thought to be involved in photosynthetic acclimation to help plants cope with environmental stress[29]. OsCyP2-P, localized in both the cytosol and nucleus, is upregulated in response to salt stress, and the ectopic expression of OsCyP2-P imparts salt stress tolerance via ROS scavenging and ion homeostasis[30]. GhCyp1 expression was higher in the roots and stems, and overexpression of GhCyp1 conferred higher tolerance to salt stress in Pseudomonas syringae pv. tabaci infection[31]. CcCYP is predominantly localized in the nucleus, and transgenic plants exhibit high tolerance to major abiotic stresses, as evidenced by increased chlorophyll levels, biomass, and plant survival[32]. In particular, transgenic plants display higher Na (+) ion accumulation to maintain ion homeostasis in the roots under salt stress[32].

    • Alfalfa (M. sativa L.) is a perennial forage crop planted worldwide that has a well-developed root system and salt tolerance. In this study, we identified nine MsCYP genes thought to play vital roles in the response to salt stress that were highly expressed during salt stress and decreased during recovery. Additionally, MsCYP273 overexpression plants showed stronger resistance to NaCl stress than wild-type plants. In summary, the identification of these MsCYPs provides a vital foundation for their possible functions in stress breeding of alfalfa. Further studies are needed to explore the functional relevance of similar genes and confirm their functional characteristics under diverse conditions.

    • The authors confirm contribution to the paper as follows: study conception and design: Liu X and Ma D; data collection: Zhang X and Xue L; analysis and interpretation of results: Chen R, Ma Q, Liu X, Zhang X and Xue L; draft manuscript preparation: Liu X, Zhang X and Xue L. All authors have read and agreed to the publication of the manuscript.

    • RNA-seq data generated in this study were deposited in NCBI under a bioproject, accession numbers as follows: the three N0 biological repeats—SRR26104474, SRR26104473, and SRR26104472; the three N2 biological repeats—SRR26104471, SRR26104470, and SRR26104469; and the three NH biological repeats—SRR26104468, SRR26104467, and SRR26104466.

      • This study was supported by Ningxia Natural Science Foundation (2022AAC03007) and The Key R&D Program of Ningxia (2021BEB04075). Thanks to OmicShare, Guangzhou, China for the RNA-seq analysis. Thanks to Elsevier for language editing assistance during the preparation of this manuscript.

      • The authors declare that they have no conflict of interest.

      • # These authors contributed equally: Xinyu Zhang, Li Xue

      • Supplemental Table S1 Primer sequences used in RT-qPCR.
      • Supplemental Table S2 The nomenclature and physicochemical properties of MsCYP450 genes.
      • Supplemental Fig. S1 Synteny analysis of CYP450 genes form alfalfa with rice, Arabidopsis, soybean, and thistle alfalfa.
      • Supplemental Fig. S2 Ka and Ks analysis of plant CYP450 duplicated genes. The number (A), Ka (B), Ks (C), and Ka/Ks (D) of CYP450 duplicated genes. Segment, segmental duplicated gene pairs; tandem, tandem duplicated gene pairs; Ms-Os, CYP450 duplicated gene pairs between alfalfa and rice; Ms-At, CYP450 duplicated gene pairs between alfalfa and Arabidopsis; Ms-Gm, CYP450 duplicated gene pairs between alfalfa and soybean; Ms-Mt, duplicated gene pairs between alfalfa and thistle alfalfa.
      • Supplemental Fig. S3 Different cis-elements in the promoters of nine MsCYP genes. White squares represent the absence of a cis-element in the promoter of MsCYP genes, and the number in the square represents the number of cis-elements in the promoter.
      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
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    Zhang X, Xue L, Chen R, Ma Q, Ma D, et al. 2023. Genome-wide identification of the cytochrome P450 family and analysis of CYP regarding salt tolerance in Medicago sativa L. Grass Research 3:21 doi: 10.48130/GR-2023-0021
    Zhang X, Xue L, Chen R, Ma Q, Ma D, et al. 2023. Genome-wide identification of the cytochrome P450 family and analysis of CYP regarding salt tolerance in Medicago sativa L. Grass Research 3:21 doi: 10.48130/GR-2023-0021

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