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Genetic mapping and gene editing reveal BoAP1 as a crucial factor regulating chloroplast development in Brassica oleracea

  • # Authors contributed equally: Xinyu Zhao, Li Chen, Kaiwen Yuan

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  • Chloroplasts are crucial cellular components that plants need to carry out photosynthesis. Exploring the relevant underlying molecular regulatory mechanism could help elucidate this complex process. In this study, a spontaneous chloroplast-deficient mutant, 6-219W, exhibiting a lethal albino phenotype in curly kale (Brassica oleracea var. acephala) was identified. The number of chloroplasts in the 6-219W mutant were considerably reduced, those chloroplasts appeared crumpled, and the thylakoid membranes could not be observed under transmission electron microscopy. Genetic analysis revealed that boap1 (Brassica oleracea albino plant1), a single recessive gene, is responsible for this lethal albino trait. Fine mapping demonstrated that boap1 is located at a 300 kb interval between the InDel markers PW404 and PW406 on chromosome 8. Based on the B. oleracea genome annotation, a candidate gene, BolC08g019310.2J, was identified within the target interval. Sequence analysis revealed a 3-nucleotide (GAT) deletion in the coding sequence of BolC08g019310.2J in the 6-219W mutant, leading to the absence of the amino acid methionine at position 298 that is conserved in Arabidopsis thaliana, B. rapa, and B. oleracea. CRISPR/Cas9 technology was used to knock out the BolC08g019310.2J gene in NB11, and the generation of boap1 mutants with an albino phenotype confirmed BolC08g019310.2J as the causal gene. Subcellular localization indicated that the BoAP1 protein operates in chloroplasts. These results revealed that the BoAP1 mutation disrupted the normal development of chloroplasts in 6-219W, leading to a lethal albino phenotype. The present research lays a foundation for the in-depth study of the molecular mechanism regulating chloroplast development.
  • 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 this study.
    Supplemental Table S2 Predicted genes associated with chloroplast development in the candidate region.
    Supplemental Fig. S1 Protein sequence alignment of BoAP1 and its relatives. AT4G8590, MRL7/ECB1/SVR4/AtRCB of Arabidopsis thaliana; Boap1, mutant-type BoAP1 in 6-219W. The 298th amino acid is indicated by the red asterisk.
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  • Cite this article

    Zhao X, Chen L, Yuan K, Liu Y, Yang L, et al. 2024. Genetic mapping and gene editing reveal BoAP1 as a crucial factor regulating chloroplast development in Brassica oleracea. Vegetable Research 4: e031 doi: 10.48130/vegres-0024-0030
    Zhao X, Chen L, Yuan K, Liu Y, Yang L, et al. 2024. Genetic mapping and gene editing reveal BoAP1 as a crucial factor regulating chloroplast development in Brassica oleracea. Vegetable Research 4: e031 doi: 10.48130/vegres-0024-0030

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Genetic mapping and gene editing reveal BoAP1 as a crucial factor regulating chloroplast development in Brassica oleracea

Vegetable Research  4 Article number: e031  (2024)  |  Cite this article

Abstract: Chloroplasts are crucial cellular components that plants need to carry out photosynthesis. Exploring the relevant underlying molecular regulatory mechanism could help elucidate this complex process. In this study, a spontaneous chloroplast-deficient mutant, 6-219W, exhibiting a lethal albino phenotype in curly kale (Brassica oleracea var. acephala) was identified. The number of chloroplasts in the 6-219W mutant were considerably reduced, those chloroplasts appeared crumpled, and the thylakoid membranes could not be observed under transmission electron microscopy. Genetic analysis revealed that boap1 (Brassica oleracea albino plant1), a single recessive gene, is responsible for this lethal albino trait. Fine mapping demonstrated that boap1 is located at a 300 kb interval between the InDel markers PW404 and PW406 on chromosome 8. Based on the B. oleracea genome annotation, a candidate gene, BolC08g019310.2J, was identified within the target interval. Sequence analysis revealed a 3-nucleotide (GAT) deletion in the coding sequence of BolC08g019310.2J in the 6-219W mutant, leading to the absence of the amino acid methionine at position 298 that is conserved in Arabidopsis thaliana, B. rapa, and B. oleracea. CRISPR/Cas9 technology was used to knock out the BolC08g019310.2J gene in NB11, and the generation of boap1 mutants with an albino phenotype confirmed BolC08g019310.2J as the causal gene. Subcellular localization indicated that the BoAP1 protein operates in chloroplasts. These results revealed that the BoAP1 mutation disrupted the normal development of chloroplasts in 6-219W, leading to a lethal albino phenotype. The present research lays a foundation for the in-depth study of the molecular mechanism regulating chloroplast development.

    • Chloroplasts are vital cellular components for photosynthesis in plants and other eukaryotic photosynthetic organisms[1]. Chloroplast development is a complex process regulated by many genes and that can be divided into three stages: the replication of plastid and plastid genomes; the establishment of genetic and translational systems, during which the chloroplast genome is mainly transcribed by nucleus-encoded RNA polymerases (NEPs); and the establishment of the chloroplast photosystem, which involves the primary transcription of photosynthesis-related genes by plastid-encoded RNA polymerases (PEPs), consisting of core subunits encoded by the rpoA, rpoB, rpoC1, and rpoC2 genes in the plastid, along with other nuclear-encoded components[2,3]. Notably, mutations in the rpoA, rpoB, rpoC1, and rpoC2 genes can impair chloroplast development, leading to a mutant leaf color phenotype.

      To date, many genes related to chloroplast development — including AtPDS (Arabidopsis phytoene desaturase), a gene related to photosynthetic pigment synthesis that encodes phytoene desaturase, a key enzyme in the carotenoid synthesis pathway — have been identified[4]. The mTERF3 (mitochondrial transcription termination factor3) protein encoded by the SL1 (seedling lethal1) gene interacts with three subunits of the PEP complex, suggesting that it may be active in chloroplast gene expression, and in Arabidopsis, the sl1 mutant shows severe deficiencies in chloroplast development and photosystem assembly[5]. The OsSLC1 (Oryza sativa seedling-lethal chlorosis1) protein, which belongs to the PPR family, is preferentially expressed in chloroplasts and plays a vital role in intron splicing[6]. In asl4 (albino seedling lethality4) mutants, the transcription levels of genes related to chlorophyll biosynthesis, photosynthesis and chloroplast development are severely inhibited, and loss of ASL4 gene function can lead to chloroplast development defects and seedling death[7].

      In Arabidopsis, the chloroplast-defective mutant mrl7/ecb1/svr4/rcb exhibits albino cotyledons, cannot grow true leaves, and dies shortly after germination. The MRL7/ECB1/SVR4/RCB gene has been mapped to At4g28590 through T-DNA insertion identification and gene localization[811]. The thioredoxin-like fold protein with disulfide reductase activity encoded by MRL7/ECB1/SVR4/RCB is localized in chloroplasts and the nucleus and is a dual-target nuclear/plastidomal photosensitive pigment signaling component necessary for PEP assembly[12]. MRL7/ECB1/SVR4/RCB has not been identified in the PEP complex, but it can interact with the three PEP complex subunits TRXz, FSD2, and FSD3, which implies that it can regulate the expression of photosynthesis-associated plastid-encoded genes (PhAPGs) through interactions with the PEP complex subunits to participate in chloroplast development[10,13]. Chloroplast-deficient mutants have also been found in Brassica crops. The yellow-green leaf (yvl) mutant was isolated from Brassica napus. The causal gene BnaA03.CHLH was identified as encoding the putative H subunit of Mg-protoporphyrin IX chelatase (CHLH)[14]. Xu et al. reported a lethal mutant, 7-521Y, with cotyledon yellowing and determined that this trait was controlled by two genes. One of the candidate genes was located at the 29 kb region of chromosome C06, and BnaC06. FtsH1, which regulates the PSII repair cycle in B. napus, was confirmed to be a target gene by complementary functional verification[15]. A chlorophyll-reduced mutant (crm1) of rapeseed was generated by EMS mutation. Two target genes with single nucleotide replacements, BnaA01G0094500ZS and BnaC01G0116100ZS, were identified via BSA sequencing in crm1. These two genes encode the CHLI1 protein, which is critical for chlorophyll synthesis[16].

      In this study, a natural lethal albino mutant, 6-219W, from curly kale that exhibited yellow cotyledons and albino hypocotyls was identified. Fine mapping, functional validation, and phylogenetic analysis were performed to explore the target gene BoAP1. The BoAP1 locus was ultimately mapped between two InDel markers, PW404 and PW406, on chromosome 8, and BolC08g019310.2J, an ortholog of the AT4G28590 gene, was the candidate gene for BoAP1. The present research provides new insights into the molecular mechanism of the lethal albino phenotype and the development of chloroplasts in cole crops.

    • 6-219G is a curly kale line; its hypocotyl and leaves are green. 6-219W is a natural mutant discovered from the self-crossing of 6-219G offspring and is characterized by white hypocotyl and yellow leaves. NB11 is a cabbage inbred line with normal hypocotyls and leaves. The heterozygous genotype 6-219G was crossed with NB11 to generate F1 plants. The F2 population for genetic analysis was obtained via self-pollination of the F1 progeny.

    • Seven- to ten-day-old 6-219G and 6-219W seedlings were collected to determine the chlorophyll content. After rapid freezing, the whole seedlings (0.1 g) were ground into powder in liquid nitrogen. The powder was immersed in 10 mL of an extraction solution (anhydrous ethanol : acetone = 1:2 (v/v)) for 4 h under light-protected extraction until the tissue residue at the bottom turned white. The abovementioned samples were centrifuged at 12,000 rpm for 15 min. The supernatant was poured into a quartz colorimeter using the extraction solution as the negative control, and the absorbance of chlorophyll at 645 and 663 nm was measured using the EV-2200 spectrophotometer[17]. Three biological replicates were performed, and the data were analyzed using the following formula[18]:

      Chla=0.01×(21.2×OD6634.48×OD645)×F÷W
      Chlb=0.01×(38.2×OD6457.8×OD663)×F÷W
      Chl=0.01×(33.7×A645+13.4×A663)×F÷W

      In the above formula, F represents the dilution factor and W represents the sample weight (g).

    • The chloroplast ultrastructures of fresh cotyledons from 6-219G and 6-219W were examined via TEM. The samples were fixed in 2.5% glutaraldehyde at 4 °C for 16 h, and then dehydration in the ethanol series and embedding was carried out[19]. The samples were prepared into slides and observed on the H-7500 transmission electron microscope (Hitachi, Tokyo, Japan).

    • Whole-genome resequencing was performed for 6-219W and NB11 using the Illumina HiSeq 2500 platform (San Diego, CA, USA). After quality control, filtering, and removal of adapter contamination using Fastp (v0.21.0) software, the BWA (v0.7.12) software was used to align the resequencing data to the Brassica oleracea cv. JZS V2.0 reference genome (http://brassicadb.cn)[20,21]. Subsequently, SNP and InDel variations were investigated by employing the GATK (v4.x) toolkit[22]. Finally, 117 InDel primers were designed on primer3 (v4.1.0) to be uniformly distributed on the nine chromosomes, with each InDel primer being spaced 3–5 Mb apart[23]. The design principles of the primers were as follows: Tm of 52–60 °C, GC content of 40%–50%, and amplification length of 100–250 bp[24]. All primers used in the present research are listed in Supplemental Table S1. Total genomic DNA was extracted from the two parental lines, the F2 population, and the transgenic plants using the improved CTAB method[25,26].

    • Sixty-six specific polymorphic markers between the parents, 6-219W and NB11 were identified from the initial 117 genome-wide markers. Both parents and 30 F2 individuals with mutant phenotypes were used to preliminarily identify linked markers. The markers at both ends of the linked interval were used to screen 96 recessive single plants to verify whether the markers were linked. Subsequently, more linked markers were developed within the linked interval, which was used for genotyping all F2 individuals.

    • A BLASTP search in the Ensembl Plants database (https://plants.ensembl.org/) was performed to download other homologous protein sequences with the BoAP1 sequence as the query. The above proteins were aligned using DNAMAN version 9.0 to display the conserved domains. A neighbor-joining phylogenetic tree (1,000 bootstrap replications) was constructed using MEGA11.0 software, with the protein sequences aligned using ClustalW.

    • CRISPR/Cas9 target prediction was carried out by employing an online website (http://crispor.tefor.net/) to select two targets located in the first exon of the BoAP1 gene: sgRNA1 (AGAACTCCGATGCTTCACACCGG) and sgRNA2 (GTCCCGCGTAAACCGAAGCGCGG). The purified fragments were cloned and inserted into zmplCas9i (derived from PC1300) using T4 DNA ligase to construct the pCas9-BoAP1 editing vector. After the successful construction of pCas9-BoAP1 was confirmed by sequencing, the vector was transformed into the recipient material NB11 using Agrobacterium-mediated methods[27].

    • To identify the subcellular localization of the BoAP1 protein, the full-length coding sequence of BoAP1 without a stop codon was ligated into the linear vector pBWA(V)HS-ccdb-GLosgfp after double enzyme digestion. After confirming the successful construction of the vector, the plasmid was introduced into tobacco leaves through Agrobacterium-mediation protocols. Then, the tobacco plants were cultured under weak light for 2 d and observed for GFP fluorescence using a laser confocal scanning microscope (Nikon C1, Japan).

    • Total RNA from 7-day-old 6-219W and 6-219G plants was extracted using the TIANGEN RNAprep Pure Plant Kit (Tiangen, Beijing, China). The qRT-PCR system was prepared with the Taq Pro Universal SYBR qPCR Master Mix Kit (Vazyme, Nanjing, China) and amplified in the Bio-Rad CFX96TM Real-Time System (Bio-Rad, Hercules, CA, USA). The relative expression of BoAP1 in the samples was calculated according to the 2−ΔΔCᴛ method, and all experiments were repeated three times for analysis[28].

    • The spontaneous albino mutant was derived from curly kale. Phenotypic observation showed that the hypocotyl of the 6-219W mutant was white, the cotyledon was yellow, and the life cycle was incomplete, with a growth period of only 7–14 d (Fig. 1ac). The chloroplasts in the 6-219G contained a complete membrane structure and stacked thylakoids under transmission electron microscopy (TEM) (Fig. 1d, e). However, the number of chloroplasts in the 6-219W mutant was considerably reduced, the plastoglobuli were on the contrary, and the thylakoid membranes could not be observed (Fig. 1f, g). The chlorophyll a and chlorophyll b levels were significantly lower in the 6-219W mutant than in the 6-219G treatment group (Fig. 1h).

      Figure 1. 

      Phenotypic characterization of wild-type 6-219W and mutant 6-219G. (a) 7-day-old seedlings of 6-219W and 6-219G. (b), (c) 10-day-old seedlings of 6-219W and 6-219G. The red arrows point to 6-219W. (d), (e) Ultrastructure of chloroplasts in 6-219G. (f), (g) Ultrastructure of chloroplasts in the albino 6-219W. Transmission electron microscopy was carried out on 7-day-old leaves of 6-219W and 6-219G. (h) Chlorophyll a (Chla), chlorophyll b (Chlb), and total chlorophyll (Chl) content in the 6-219W and 6-219G groups. The asterisk represents the magnitude of the difference: *** p < 0.001 and **** p < 0.0001. Scale bars: (a)−(c) 1 cm; (e), (g) 10 μm; (f), (h) 1 μm.

      Since 6-219W is a lethal albino mutant, heterozygous 6-219G was crossed with NB11, a wild-type cabbage inbred line, to generate the F1 progeny and F2 mapping population. F1 individuals exhibit normal phenotypes. Among the 2,580 plants in the F2 population, there were 652 albino seedlings and 1,928 normal seedlings. The genetic separation ratio between the lethal albino seedlings and the normal individuals was 3:1 (χ2 = 0.101 < χ20.05 = 3.841), demonstrating that a single recessive gene causes this lethal albino trait.

    • Based on the genome comparison between 6-219W and NB11, 117 InDel markers evenly distributed across nine chromosomes of cabbage were designed to identify polymorphisms between the parents. A total of 66 markers differed between the two parents. First, 30 albino-lethal individuals were screened with the above 66 differential markers, and we found that the region linked to the albino trait was 12.95 Mb (between InDel markers PW81 and PW304) on chromosome 8 (Fig. 2a). Subsequently, 405 F2 recessive plants with a lethal albino phenotype were mapped based on ten polymorphic InDel markers, indicating a 0.92 Mb region (between the markers PW373 and PW374) on chromosome 8 (Fig. 2a). Finally, eight polymorphic markers were developed 1,642 F2 recessive individuals were selected for fine mapping. The boap1 locus was ultimately identified in an approximately 300 kb region (between the InDel markers PW404 and PW406) on chromosome 8 (Fig. 2a). Three genes related to chloroplast development were determined within the 300 kb candidate region (Supplemental Table S2).

      Figure 2. 

      Map-based cloning of BoAP1. (a) Fine mapping of BoAP1 with InDel markers. The solid red rectangle represents the position of BolC08g019310.2J. (b) The schematic diagram depicts the exons (solid black boxes) and introns (black lines) of BoAP1. The nucleotide sequence and sequence mapping show the point mutation in 6-219W. The black frame represents the location of the boap1 mutation.

      The subsequent sequencing analysis of the BolC08g019310.2J gene indicated homozygous deletions of three nucleotides (GAT) in the fourth exon in the 6-219W mutant. In comparison, there were homozygous insertions of three nucleotides (GAT) or heterozygous genotypes in the 6-219G individuals, corresponding to the phenotype (Fig. 2b). The putative protein sequence in the 6-219W mutants had a deletion of a Met at the 298th amino acid position (Supplemental Fig. S1). Protein sequence alignment and conserved motif analysis indicated that the mutation site was located between motif 9 and motif 5 and was conserved in cabbage, Arabidopsis, and Chinese cabbage (Supplemental Fig. S1). Moreover, BolC08g019310.2J shared 77% amino acid sequence identity with AT4G28590, a plastidial phytochrome signaling component in the chloroplast[8]. The above results indicated that deleting the 298th amino acid M might lead to the loss of BolC08g019310.2J function. Therefore, BolC08g019310.2J was designated BoAP1 in this study.

    • To elucidate the phylogenetic relationship between BoAP1 and other homologous proteins, the homologous protein sequences of the Brassicaceae, Solanaceae, and Leguminosae families, among others, were downloaded through BLASTP and then a phylogenetic evolutionary tree was reconstructed (Fig. 3b). The results showed that BoAP1 is most closely related to AT4G28590 in Arabidopsis, indicating that BoAP1 plays an essential and conserved role in chloroplast development.

      Figure 3. 

      Gene expression patterns of BoAP1. (a) Expression levels of BoAP1 in the 6-219G and 6-219W. The asterisk represents the magnitude of the difference (***, p < 0.001). (b) Phylogenetic analysis of BoAP1 and its related proteins.

    • According to previous reports, AT4G28590 encodes a protein localized in the chloroplast[8], so it was speculated that the protein encoded by BoAP1 is also expressed in the chloroplasts. To verify this prediction, a BoAP1-GFP fusion protein expression vector was constructed and transferred into tobacco leaf cells for transient expression. Laser scanning confocal microscopy showed that the fluorescence signal of the BoAP1-GFP fusion protein overlapped with chloroplast autofluorescence, while the GFP green fluorescence of the empty vector was detected in the nucleus and the cytoplasm (Fig. 4). Taken together, these results indicated that BoAP1 is localized to chloroplasts.

      Figure 4. 

      Subcellular localization analysis of BoAP1. The tobacco leaves were transformed with the empty GFP vector and BoAP1-GFP fusion protein. From left to right: fluorescence of the empty GFP vector and BoAP1; spontaneous fluorescence in chloroplasts; bright-field image; merged image of the first three images. Scale bars = 20 μm.

      Quantitative real-time PCR (qRT-PCR) was performed to compare the expression of the BoAP1 transcript in 6-219G and 6-219W. The results indicated that the transcription of BoAP1 was significantly downregulated in the 6-219W mutants, inferring that the expression of BoAP1 is closely related to the albino phenotype (Fig. 3a).

    • To confirm whether a functional loss of BoAP1 causes the albinism phenotype, we constructed a pCas9-BoAP1 genome-editing vector with two sgRNAs targeting exon 1 of BoAP1 (Fig. 5a). The pCas9-BoAP1 vector was transferred into wild-type NB11 explants via the Agrobacterium tumefaciens-mediated genetic transformation method. Twenty positive lines were generated by amplifying the Hyg gene. Sanger sequencing analysis of the amplified BoAP1 gene revealed that eight T0-positive transgenic lines were edited and exhibited an albino phenotype (Fig. 5b, c).

      Figure 5. 

      Functional confirmation of BoAP1. (a) Schematic diagram of the pCas9-BoAP1 genome-editing vector targeting BoAP1. Hyg, hygromycin resistance gene; U6, Arabidopsis U6 promoter. (b) Phenotypes of the WT, boap1-1, and boap1-2. Scale bar: 0.5 cm. (c) Sequence mapping of the WT, boap1-1, and boap1-2 on the target region.

    • Chloroplasts are vital organelles plants need to carry out photosynthesis. They provide organic material and energy for plants and are essential for the growth and development of green plants[29]. Through the in-depth study of chloroplasts, many chloroplast-deficient mutants and corresponding causal genes have been discovered in A. thaliana, Oryza sativa, B. napus, and others, revealing details of the chloroplast growth and development process[5,14,15,30,31]. In this study, a chloroplast-deficient mutant, 6-219W, with yellow cotyledons and white hypocotyls that has an abnormal growth period and survives for only 7–10 d was identified. Its phenotype resembles that of the rapeseed line 7-521Y[15]. An F2 population was used to fine-map the target gene to a 300 kb region on chromosome 8, and through gene sequencing, sequence analysis, and functional verification, it was found that BolC08g019310.2J is a strong candidate for being the gene responsible for the albino trait.

      Leaf color is an important target trait in vegetable genetics and breeding, which directly affects its commodity value. Chloroplasts are vital for the shelf life of leafy vegetables. Gene mutations such as BoYgl-2 and BrCAO cause yellow leaf, affecting their yield and commercial value, and shortening their shelf life[32,33]. Mutation in the BrNYM1 gene can maintain leaves greenness of Chinese cabbage during the aging process and extend their shelf life[34]. The full-length introduction of the BoAP1 homologous gene AtMRL7 driven by the 35S promoter into the mrl7 Arabidopsis albino mutant increases the chloroplast number of the mutant and restores the normal phenotype[8]. Therefore, it was speculated that overexpression of the BoAP1 gene might increase the number of chloroplasts and prolong the life and shelf time.

      The CRISPR/Cas9 gene editing system is already widely used in plants such as rice, maize, Arabidopsis, tobacco, tomatoes, and cabbage. The lethal albino gene has been successfully applied to test the efficiency of the CRISPR/Cas9 gene editing system because its editing efficiency can be easily observed during the seedling or sprouting stage. CRISPR/Cas9 has been used to knock out the OsPDS gene in rice, and an albino phenotype was observed in the T0 generation[35]. Agrobacterium-mediated CRISPR/Cas9 technology was previously used to knock out the AtPRPL18 and OsPRPL18 genes, and the atprpl18 and osprpl18 mutants exhibited albino seedling phenotypes[31]. Pan et al.[36] and Ma et al.[37] targeted mutations in the PDS homologous genes in tomatoes and cabbage, respectively, and found a high mutation frequency in the T0 transgenic plants. In the present research, a CRISPR/Cas9 editing vector was used to knock out the BoAP1 gene, and the resulting boap1 mutant exhibited a typical lethal albino phenotype. We believe that this gene may be similar to the PDS gene, and the efficiency of various editing systems can be tested by knocking out the homologous genes of BoAP1 in crops.

      The PEP complex contains plastid-encoded rpo subunits and other nucleus-encoded proteins, among which FSD2, FSD3, TRXz and FLN1 may play an important role in the redox-mediated regulation of chloroplast development[3840]. FSD2 and FSD3 are superoxide dismutases[41]. Loss of FSD2 or FSD3 function impairs the growth of Arabidopsis plants and leads to leaf albinism. The fsd2 mutant appears increased superoxide yield, decreased chlorophyll content, and a decreased CO2 assimilation rate, while the fsd3 mutant does not survive beyond the seedling stage[42]. Through CRISPR/Cas9-directed mutagenesis of OsFLN1, a severe lethal albino phenotype was observed in mutants and OsFLN1 was found to regulate the transcription of PEP-related genes and chloroplast growth and development by interacting with OsTRXz[43]. OsTRXz, a chloroplast thioredoxin reductase is indispensable for maintaining the complete structure of the PEP complex and regulating chloroplast biosynthesis. In the present study, the homologous gene BoAP1 of AtMRL7/ECB1/SVR4/RCB was cloned in curly kale. Previous studies have reported that AtMRL7/ECB1/SVR4/RCB can interact with TRXz, FSD2, and FSD3, and that compared to those of untreated RNAi lines, AtMRL7-RNAi lines treated with appropriate concentrations of ROS-related reagents show greater chlorophyll content and a significant recovery of the pale green phenotype, indicating that MRL7/ECB1/SVR4/RCB is involved in the redox-mediated regulation of chloroplast development[10,13]. Therefore, to further explore the regulatory network of the BoAP1 protein, it was hypothesized that BoAP1 interacts with TRXz, FSD2, and FSD3 in Brassica plants and that different concentrations and types of ROS can also restore the green phenotype of 6-219W or boap1 mutants.

    • In conclusion, a spontaneous chloroplast-deficient mutant, 6-219W was identified. A single recessive gene, BoAP1, is responsible for this lethal albino trait. Fine mapping indicated that BolC08g019310.2J was identified as the candidate gene. Through CRISPR/Cas9 technology, it was demonstrated that the BoAP1 mutation disrupted the normal development of chloroplasts in 6-219W, leading to a lethal albino phenotype. The present research provides a basis for the in-depth study of the molecular mechanism regulating chloroplast development.

    • The authors confirm contribution to the paper as follows: study conception and design: Lv H; performing experiments: Zhao X, Yuan K; writing and revising manuscript: Zhao X, Chen L, Fujimoto R; data analyses: Liu Y; assisting experiments: Yang L, Zhuang M, Zhang Y, Wang Y, Ji J. All authors reviewed the results and approved the final version of the manuscript.

    • All data generated or analyzed during this study are included in this published article and its supplementary information files.

      • This work was supported by grants from the National Key R&D Program of China (2023YFD1201501), the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS), and China Agriculture Research System of MOF and MARA (CARS-23).

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

      • # Authors contributed equally: Xinyu Zhao, Li Chen, Kaiwen Yuan

      • Supplemental Table S1 Primer sequences used in this study.
      • Supplemental Table S2 Predicted genes associated with chloroplast development in the candidate region.
      • Supplemental Fig. S1 Protein sequence alignment of BoAP1 and its relatives. AT4G8590, MRL7/ECB1/SVR4/AtRCB of Arabidopsis thaliana; Boap1, mutant-type BoAP1 in 6-219W. The 298th amino acid is indicated by the red asterisk.
      • Copyright: © 2024 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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    Zhao X, Chen L, Yuan K, Liu Y, Yang L, et al. 2024. Genetic mapping and gene editing reveal BoAP1 as a crucial factor regulating chloroplast development in Brassica oleracea. Vegetable Research 4: e031 doi: 10.48130/vegres-0024-0030
    Zhao X, Chen L, Yuan K, Liu Y, Yang L, et al. 2024. Genetic mapping and gene editing reveal BoAP1 as a crucial factor regulating chloroplast development in Brassica oleracea. Vegetable Research 4: e031 doi: 10.48130/vegres-0024-0030

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