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Prunus mume genome research: current status and prospects

  • # Authors contributed equally: Dongqing Fan, Runtian Miao

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  • Mei (Prunus mume) is an excellent garden tree highly praised in China, possessing both ornamental and cultural values. Breeding Mei with distinctive characteristics and high resistance has become a long-term goal to meet the visual and spiritual demands in the new era. With the rapid development of biotechnology, researchers have successively completed the whole genome sequence and resequencing of Mei, and continue to employ advanced techniques to investigate the formation mechanisms of important ornamental traits and stress resistance traits in Mei. Thus, the groundwork has been established for achieving the breeding objectives. In this article, we provide an overview of the development and expansion of genome projects over the past decade, including whole-genome sequencing, resequencing, and genetic mapping. We further present a concise summary of the research progress made in understanding major ornamental traits and cold resistance traits. These accomplishments hold great promise for significantly enhancing the efficiency of Mei and further realizing breeding goals.
  • Bletilla Rchb. f. is one of the most economically valuable groups of orchids in the world. Due to its ornamental significance, the genus Bletilla occupies an important place in the worldwide horticultural market. Furthermore, in China, Japan, South Korea, and other Asian countries, it is highly valued for its medicinal use[1].

    There are eight species in the genus Bletilla, including Bletilla chartacea (King & Pantl.) Tang & F.T. Wang, Bletilla cotoensis Schltr., Bletilla foliosa (King & Pantl.) Tang & F.T. Wang, Bletilla formosana Schltr., Bletilla guizhouensis J. Huang & G.Z. Chen, Bletilla morrisonensis Schltr., Bletilla ochracea Schltr., and Bletilla striata Rchb.f.[2,3]. The distribution area spans from northern Myanmar in Asia to Japan via China[4]. Five species are native to China, namely, B. foliosa, B. formosana, B. guizhouensis, B. ochracea, and B. striata. In China, people have assigned various names to Bletilla based on its morphology and efficacy, such as baiji (白及/白芨), baigen (白根), baige (白给), baijier (白鸡儿), baijiwa (白鸡娃), diluosi (地螺丝), gangen (甘根), junkouyao (皲口药), lianjicao (连及草), and yangjiaoqi (羊角七)[5]. These diverse appellations highlight the importance of this genus in Chinese folk biological culture.

    The medicinal material known as 'baiji' in traditional Chinese medicine (TCM) is usually the dried tuber of B. striata, which is also the authentic product included in the Chinese Pharmacopoeia[6]. According to the Chinese Pharmacopoeia (2020), TCM baiji is sliced, dried, and crushed into a powder that can be used topically or internally, with a recommended dosage of 3–6 g at a time, offering astringent, hemostatic, detumescence, and myogenic effects. It is often used for conditions such as hemoptysis, hematemesis, traumatic bleeding, sores, and skin chaps[7]. Although only B. striata is the authentic product of TCM baiji, the other four Bletilla species native to China are also used as substitutes, and this practice is widespread[8].

    Modern research indicates that Bletilla contains a variety of chemical components, including benzol, dihydrophenanthrene, phenanthrene, and quinone derivatives. These components confer pharmacological effects on Bletilla, such as hemostasis, anti-tumor activity, and promotion of cell growth[9]. Due to its outstanding medicinal value, Bletilla can be found in nearly every corner of the traditional medicine market (Fig. 1). However, habitat destruction and uncontrolled mining have led to a significant reduction in the native populations of Bletilla, making its protection an urgent priority. Therefore, this paper provides a comprehensive review of relevant research up to August 2023, covering botanical characteristics, resource distribution, ethnobotanical uses, chemical components, pharmacological effects, clinical applications, and safety evaluations of Bletilla. The aim is to raise awareness and promote the protection and sustainable use of this genus.

    Figure 1.  Varieties of Bletilla at the traditional March Medicinal Market in Dali, Yunnan, China.

    The morphology of different Bletilla species is highly similar. The primary taxonomic feature distinguishing each species is the characteristics of the flower, particularly the lip of the flower, including its size, shape, and the number and shape of longitudinal ridges on the lip plate (Table 1, Fig. 2)[1014].

    Table 1.  The morphological differences among five species of Bletilla plants native to China.
    Morphological featureBletilla striataBletilla formosanaBletilla ochraceaBletilla foliosaBletilla guizhouensis
    Plant height (cm)18−6015−8025−5515−2045−60
    Rhizome shapeCompressedCompressedSomewhat compressedSubgloboseCompressed
    Rhizome diameter (cm)1−31−2About 21−1.53−4
    Stem characteristicsStoutEnclosed by sheathsStoutStout, shortThin
    Leaf shapeNarrowly oblongLinear-lanceolateOblong-lanceolateElliptic-lanceolateNarrowly lanceolate
    Leaf size (cm)8−29 × 1.5−46−40 × 0.5−4.58−35 × 1.5−2.85−12 × 0.8−325−45 × 1.2−4.5
    Flower colorPurplish red or pinkPale purple or pinkYellowPale purpleDeep purple
    Flower sizeLargeMediumMediumSmall to mediumLarge
    Inflorescence structureBranched or simpleBranched or simpleSimpleSimpleBranched
    Pedicel and ovary length (mm)10−248−12About 187−913−17
    Sepal shapeNarrowly oblongLanceolateLanceolateLinear-lanceolateOblong-elliptic
    Petal shapeSlightly larger than sepalsSlightly narrower than sepalsObliqueLanceolateOblong-elliptic
    Lip shapeObovate-ellipticBroadly ellipticNarrowly rhombic-obovateNarrowly oblongNarrowly oblong
    Lip colorWhite with purplish veinsWhitish to pale yellow with small dark purple spotsWhitish to pale yellow with small dark purple spotsWhite with purplish spots and purple edgeWhite with deep purple edge
    Number of lip Lamellae5 lamellae5 undulate lamellae5 longitudinal lamellae3 fimbriate lamellae7 longitudinal lamellae
    Column characteristicsSubterete, dilated towards apexSubterete, dilated towards apexSlender, dilated towards apexCylindric, dilated towards apexSuberect, with narrow wings
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    Figure 2.  (a)−(d) Bletilla striata (Thunb. ex Murray) Rchb. f. (e)−(h) Bletilla formosana (Hayata) Schltr. (i)−(l) Bletilla ochracea Schltr. (m), (n) Bletilla sinensis (Rolf) Schltr. (o), (p) Bletilla guizhouensis Jie Huang & G.Z. Chen (Photographed by Wang Meina, Zhu Xinxin, and He Songhua).

    The flowers of B. striata are large and purplish-red or pink, with narrowly oblong sepals and petals measuring 25−30 mm in length and 6−8 mm in width. They have acute apices, nearly as long as the sepals and petals. The lip is obovate or elliptic, predominantly white with purplish-red coloration and purple veins, measuring 23−28 mm in length, slightly shorter than the sepals and petals. The lip disc exhibits five longitudinal folds extending from the base to near the apex of the middle lobe, with waviness occurring only above the middle lobe[11]. In China, B. striata is found in regions such as Anhui, Fujian, Guangdong, Guangxi, Gansu, Guizhou, Hubei, Hunan, Jiangsu, Jiangxi, Shaanxi, Sichuan, and Zhejiang. It also occurs in the Korean Peninsula and Japan, thriving in evergreen broad-leaved forests, coniferous forests, roadside grassy areas, or rock crevices, at altitudes ranging from 100−3,200 m[12].

    B. ochracea's flowers are medium to large, featuring yellow or yellow-green exteriors on the sepals and petals, while the insides are yellow-white, occasionally nearly white. The sepals and petals are nearly equal in length, oblong, measuring 18−23 mm long and 5−7 mm wide, with obtuse or slightly pointed apices, often adorned with fine purple spots on the reverse side. The lip is elliptic, typically white or light yellow, measuring 15−20 mm in length and 8−12 mm in width, with three lobes above the middle. The lip disc is characterized by five longitudinally ridged pleats, with undulations primarily occurring above the middle lobe[13]. B. ochracea is native to southeastern Gansu, southern Shaanxi, Henan, Hubei, Hunan, Guangxi, Guizhou, Sichuan, and Yunnan, thriving in evergreen broad-leaved forests, coniferous forests, or beneath shrubs, in grassy areas or alongside ditches at altitudes ranging from 300−2,350 m[14].

    B. formosana's flowers come in shades of lavender or pink, occasionally white, and are relatively small. The sepals and petals are narrowly oblong, measuring 15−21 mm in length and 4−6.5 mm in width, and are nearly equal in size. The sepals have subacute apices, while the petal apices are slightly obtuse. The lip is elliptic, measuring 15−18 mm in length and 8−9 mm in width, with three lobes above the middle. The lip disc exhibits five longitudinal ridge-like pleats, which are wavy from the base to the top of the middle lobe[15]. B. formosana is indigenous to southern Shaanxi, southeastern Gansu, Jiangxi, Taiwan, Guangxi, Sichuan, Guizhou, central to northwest Yunnan, southeast Tibet (Chayu), and Japan. It is typically found in evergreen broad-leaved forests, coniferous forests, road verges, valley grasslands, grassy slopes, and rock crevices, at altitudes ranging from 600−3,100 m[16].

    The flowers of B. foliosa are small and lavender, with white sepals and petals featuring purple apices. The sepals are linear-lanceolate, measuring 11−13 mm in length and 3 mm in width, with subacute apices. The petals are lanceolate, also measuring 11−13 mm in length and 3 mm in width, with acute apices. The lip is white, oblong, adorned with fine spots, and features a purple apex. It measures 11−13 mm in length and 5−6 mm in width, tapering near the base and forming a scaphoid shape. The lip is anteriorly attenuated, unlobed, or abruptly narrowing with inconspicuous three lobes and exhibits fringe-like fine serrations along the edge. Three longitudinal ridge-like pleats are present on the upper lip disc[17]. B. foliosa typically grows on hillside forests, with its type specimen collected from Mengzi City, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province, China[17].

    B. guizhouensis is a recently discovered species in Guizhou, China. In terms of shape, B. guizhouensis closely resembles B. striata, but it can be distinguished by its ovate-oblong buds, oblong dorsal sepals, obovate lips, and middle lobes of the lips, which are oval in shape. The disc of B. guizhouensis features seven distinct longitudinal lamellae, setting it apart from other known Bletilla species and establishing it as a distinct species[2]. Presently, B. guizhouensis has only been found in Guizhou, China, primarily thriving in evergreen broad-leaved forests at altitudes ranging from 900−1,200 m[3].

    Understanding the morphology, habitat, and distribution of Bletilla species is crucial for the conservation and propagation of these resources. To effectively implement plant conservation and breeding programs, a comprehensive understanding of the specific morphological characteristics, growth environments, and native habitats of these plants is essential, as without this knowledge, effective results cannot be achieved.

    The ethnobotanical uses of Bletilla worldwide primarily fall into two categories: ornamental and medicinal purposes. Bletilla orchids, renowned for their striking and distinct flowers, are commonly cultivated for ornamental purposes across many countries[18]. Valued for their aesthetic appeal, these orchids are frequently grown in gardens and utilized as potted plants. Among the various cultivars, B. striata stands out as the most favored choice for ornamental horticulture due to its ease of cultivation and adaptability to diverse climates[19,20].

    Contrastingly, in select Asian countries, Bletilla assumes a crucial role as a medicinal plant. For instance, influenced by TCM, the tuber of Bletilla also serves as a crude drug for hemostatic and anti-swelling purposes in Japan[21]. Likewise, traditional Korean medicine, deeply rooted in TCM principles, extensively documents the versatile use of Bletilla in addressing issues such as alimentary canal mucosal damage, ulcers, bleeding, bruises, and burns[22]. In Vietnam, Bletilla has been used as a medicinal herb for treating tumors and skin fissures, aligning with practices observed in the ethnic communities of southwest China[23].

    In China, Bletilla boasts a longstanding medicinal history, with numerous classical ancient Chinese medicine books containing detailed records of its medicinal applications[2432]. Even in contemporary society, many ethnic groups residing in mountainous areas in China continue to uphold the traditional medical practice of using Bletilla medicinally[31].

    In ancient Chinese medical literature, detailed records of Bletilla's morphology can be traced back to the late Han Dynasty, around 200 AD[24]. The Mingyi Bielu, a historical source, documented, 'Bletilla grows in the valley, with leaves resembling those of Veratrum nigrum L., and its root is white and interconnected. The ideal time for harvesting is September'. As awareness of the medicinal significance of Bletilla grew, successive dynastic-era Chinese medical texts consistently included descriptions of Bletilla's morphology (Table 2). In the Ming Dynasty, Li Shizhen compiled these earlier accounts of Bletilla's plant characteristics in his work, the Compendium of Materia Medica. He even provided an illustrative depiction of this plant genus (Fig. 3)[25].

    Table 2.  Morphological description of the plants belonging to Bletilla in the ancientChinese medicinal books.
    Dynasty (Year)TitleAuthorOriginal ChineseEnglish translation
    Late Han
    (184−220 AD)
    Mingyi Bielu/白给生山谷, 叶如藜芦,
    根白相连, 九月采
    Bletilla grows in the valley, with leaves like Veratrum nigrum L., root is white and connected. September is the time for harvesting.
    Wei-Jin period
    (220−420 AD)
    WuPu BencaoWu Pu白根, 茎叶如生姜, 藜芦,
    十月花, 直上, 紫赤色,
    根白连, 二月, 八月, 九月采
    Bletilla, stems and leaves like Zingiber officinale Roscoe and V. nigrum. It blooms in October and is purple and red, the inflorescence is vertical and upward. The roots are white and connected. It can be dug in February, August, and September.
    the Northern and Southern
    (420−589 AD)
    Bencao JizhuTao Hongjing近道处处有之, 叶似杜若,
    根形似菱米, 节间有毛
    It is everywhere near the road. The leaves are like Pollia japonica Thunb. The roots are like the fruit of Trapa natans L., and internode are many fibrous roots.
    Tang
    (618−907 AD)
    Su Jing, Zhangsun Wuji, etcTang materia medica生山谷, 如藜芦, 根白连, 九月采Born in the valley, with leaves like V. nigrum, root is white and connected. September is the time for harvesting.
    Song
    (960−1279 AD)
    Su SongCommentaries on the Illustrations白芨, 生石山上。春生苗,
    长一尺许, 似栟榈及藜芦,
    茎端生一台, 叶两指大, 青色,
    夏开花紫, 七月结实, 至熟黄黑色。
    至冬叶凋。根似菱米, 有三角白色, 角端生芽。二月, 七月采根
    Bletilla grow on the stone hill. It sprouts in spring and grows about a foot long. The seedlings are like Trachycarpus fortunei (Hook.) H. Wendl. and V. nigrum. The leaves are two finger-size. In summer, it blooms purple flowers and bears fruit in July. The ripe fruit is yellow-black. The leaves wither in winter. The root is like the fruit of T. natans, with three corners, white, and sprouting at the corners. The roots are dug in February and July.
    Ming
    (1368−1644 AD)
    Li ShizhenCompendium of Materia Medica一棵只抽一茎, 开花长寸许,
    红紫色, 中心如舌, 其根如菱米,
    有脐, 如凫茈之脐,
    又如扁扁螺旋纹, 性难干
    Only one stem per herb. The flower is more than one inch long, red and purple, and the center resembles a tongue. Its root is similar to the fruit of T. natans, possessing an umbilicus akin to that of Eleocharis dulcis (N. L. Burman) Trinius ex Henschel. It has spiral veins and is challenging to dry.
    −, Anonymous.
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    Figure 3.  Bletilla in Compendium of Materia Medica.

    Generally, ancient Chinese medical texts did not make clear distinctions between different Bletilla species. They collectively referred to plants with similar morphological traits as 'baige', 'baigen', 'baiji', 'gangen', 'lianjicao', or 'ruolan'. However, through textual analysis, it has been established that the descriptions of Bletilla in ancient texts before the Ming Dynasty largely align with Bletilla striata in terms of plant height, pseudo-bulb shape, leaf morphology, flower and fruit colors, and other characteristics. While the Bletilla portrayed in attached images may not precisely match B. striata in terms of morphology, considering the textual descriptions, it generally corresponds with B. striata. In writings from the Ming Dynasty and later periods, more specific descriptions of Bletilla emerged, encompassing details about its vascular arrangement, inflorescence, and flower structure, which consistently align with B. striata. Consequently, researchers have corroborated that the original plant of Bletilla described in ancient texts is Bletilla striata[24,33].

    According to the ancient Chinese medicinal books, Bletilla was used to treat a wide variety of conditions, including coughing, bruising, and bleeding, but their most mentioned use in ancient Chinese texts is for skin whitening and freckle removal[25]. Since ancient times, Bletilla species have been used consistently for skin care and whitening, and there are many well-known skincare products related to Bletilla. These Chinese formulae with Bletilla are similar to modern facial masks, face creams, facial cleanser, hand creams and other skin care products[26].

    For example, a prescription for 'facial fat (面脂)' in Medical Secrets from the Royal Library (752 AD) is made by boiling Bletilla with other traditional ingredients, and is applied to the face, resulting in skin whitening, freckle and wrinkle removal[27]. The 'Angelica dahurica cream (白芷膏)' in the General Medical Collection of Royal Benevolence (1111−1125 AD) is reputed to whiten facial skin through a seven-day treatment regiment, and contains Bletilla as the main botanical ingredient along with Angelica dahurica[28]. Jingyue Quanshu (1563-1640 AD) also contains a prescription called 'Yurong powder (玉容散)' for facial skin care. 'Yurong powder' is made of a fine powder of Bletilla, Nardostachys jatamansi (D. Don) DC., Anthoxanthum nitens (Weber) Y. Schouten & Veldkamp and other herbs[29]. Washing the face with Yurong powder in the morning and evening every day is said to make a person's face white without blemishes (Fig. 4)[29].

    Figure 4.  Yurong powder made of Bletilla and other traditional Chinese medicines in Jingyue Quanshu.

    In addition, in ancient Chinese medicine texts, Bletilla is also a well-known medicine for treating hematemesis, hemoptysis and bruises[23]. According to Shennong's Classic of Materia Medica (25−220), grinding the white fungus into fine powder and taking it after mixing with rice soup can be effective for treating lung damage and hematemesis[30]. Among the Prescriptions for Universal Relief (1406), 18 traditional Chinese medicines, such as Bletilla, are used to make 'snake with raw meat cream', which is said to be useful to treat carbuncles and incised wounds[31]. There is also a record of Bletilla powder treating lung heat and hematemesis in the Collected Statements on the Herbal Foundation (1624)[32].

    In ancient Chinese medicinal texts, most Bletilla are said to be useful for lung injury and hemoptysis, epistaxis, metal-inflicted wounds, carbuncles, burns, chapped hands and feet, whitening and especially for skin care. In the ancient medicinal texts, Bletilla is used alone or mixed with other traditional Chinese medicines. It is usually used in the form of a powder. The various medicinal effects of Bletilla described in these ancient texts suggest the great potential of this genus in clinical application, especially in the market of skin care products and cosmetics.

    As a skin care herb praised by ancient medical classics, 11 ethnic minorities in China, such as Bai, Dai, De'ang, Jingpo, Lisu, Miao, Mongolian, Mulao, Tu, Wa, and Yi still retain the traditional habit of using Bletilla for skin care in their daily life (Table 3). In addition to B. striata, B. formosana and B. ochracea are also used as substitutes. Although Chinese ethnic groups have different names for Bletilla spp., the skin care methods are basically the same. Dry Bletilla tubers are ground into a powder and applied to the skin[34], and this usage is also confirmed by the records in ancient medical texts[23, 24]. The various local names of Bletilla by different ethnic groups also indirectly suggests which ethnic groups play an important role in the traditional use. For example, Bai people called B. striata baijier (白鸡儿), goubaiyou (狗白尤), and yangjiaoqi (羊角七) (Table 3).

    Table 3.  The traditional medicinal knowledge of Bletilla in ethnic communities, China.
    Ethnic groupLatin nameLocal nameUsed partUse methodMedicinal effect
    AchangBletilla striata (Thunb. ex Murray) Rchb. F.BaijiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationTuberculosis, hemoptysis, bleeding from gastric ulcer, burns and scalds
    BaiBaijier, Goubaiyou, YangjiaoqiTuberTreatment of tuberculosis hemoptysis, bronchiectasis hemoptysis, gastric ulcer hemoptysis, hematochezia, skin cracking
    DaiYahejieTuberUsed for tuberculosis, tracheitis, traumatic injury, and detumescence
    De'angBageraoTuberTuberculosis, hemoptysis, bleeding from gastric ulcer, burns and scalds
    DongShaque, SanjueTuberTreat hematemesis and hemoptysis
    JingpoLahoiban, PusehzuotuberFor tuberculosis, bronchiectasis, hemoptysis, gastric ulcer, hematemesis, hematuria, hematochezia, traumatic bleeding, burns, impotence
    MengMoheeryichagan, NixingTuberFor tuberculosis hemoptysis, ulcer bleeding, traumatic bleeding, chapped hands, and feet
    MiaoBigou, Wujiu, SigouTuberUsed for hemoptysis of tuberculosis, bleeding of ulcer disease, traumatic bleeding, chapped hands, and feet
    MolaoDajiebaTuberTreat internal and external injuries caused by falls
    TibetanSanchabaijiTuberFresh chopped and soaked with honey; Powdered after sun-dried, then taken with honey and waterMainly used to treat cough, asthma, bronchitis, lung disease and a few gynecological diseases
    TuRuokeyeTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationTreatment of tuberculosis, hemoptysis, bloody stool, chapped skin
    WaBaijiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationFor tuberculosis, hemoptysis, gastrointestinal bleeding, scald and burn
    YaoBiegeidaiTuberTreat gastric ulcer, pulmonary tuberculosis, cough, hemoptysis, and hematemesis
    YiDaibaij, Tanimobbaili, Niesunuoqi, AtuluoboTuberTreatment of tuberculosis, hemoptysis, golden wound bleeding, burns, chapped hands and feet
    ZhuangManggounuTuberTreat stomachache and hemoptysis
    BaiBletilla formosana (Hayata) Schltr.Baijier, YangjiaoqiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationIt is used for emesis, hemoptysis due to tuberculosis, and hemoptysis due to gastric ulcer. External application for treatment of incised wound
    MiaoLianwuTuberThe effect is the same as that of B. striata
    LisuHaibiqiuTuberIt can treat tuberculosis, hemoptysis, epistaxis, golden sore bleeding, carbuncle and swelling poison, scald by soup fire, chapped hands and feet
    YiNiesunuoqi, Yeruomaoranruo, Atuluobo, Ribumama, Atuxixi, Abaheiji, Binyue, ZiyouTuberIt is used for tuberculosis, hemoptysis, traumatic injury, treatment of frostbite, burn, scald, bed-wetting of children and other diseases
    BaiBletilla ochracea Schltr.Baijier, YangjiaoqiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationFor hematemesis, epistaxis, hemoptysis due to tuberculosis, hemoptysis due to gastric ulcer; External application of golden sore and carbuncle
    MengMoheeryichagan, NixingtuberThe effect is the same as that of B. striata
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    The formation of traditional medical knowledge among Chinese people is often directly related to the specific living environment and cultural background[34]. For example, the Bai, Dai, De'ang, Jingpo, Lisu Yi, Wa and other ethnic minorities live in mountainous areas. The cold weather in winter and year-round outdoor manual work makes it difficult to maintain their skin[35, 36]. In the face of this situation, the ethnic people who are concerned about their physical appearance have long ago chosen local Bletilla species for skin care, and have handed down this tradition for many generations[34]. This important traditional skin care tradition is worthy of further in-depth study.

    The six main classes of Bletilla chemical components, phenanthrene derivatives, phenolic acids, bibenzyls, flavonoids, triterpenoids, and steroids, have been described previously. Almost three hundred compounds have been isolated from Bletilla, including 116 phenanthrene derivatives, 58 phenolic acids, 70 bibenzyls, 8 flavonoids, 24 triterpenoids and steroid and 13 other compounds (Figs 514). Chemical structures of the isolates of Bletilla species most are phenanthrene derivatives, which have been demonstrated to possess various pharmacological activities.

    The prominent opioids oxycodone, hydrocodone, naloxone, and naltrexone are all phenanthrene derivatives[37]. Currently, phenanthrene derivatives (Fig. 5, 1 to 66) were isolated from B. formosana, B. ochracea, and B. striata. In 2022, 17 phenanthrene derivatives (117) were isolated from the ethyl acetate (EtOAc) extracts of B. striata tubers[38]. Then, other phenanthrene derivatives were isolated from Bletilla, such as dihydrophenanthrenes (1841), phenanthrenes (4266), biphenanthrenes (Fig. 6, 6789), dihydro/phenanthrenes with uniquestructures (90112) and phenanthraquinones (Fig. 7, 113116). Thus far, this genus has been documented to include these compounds, which have been shown to exhibit pharmacological actions[3945].

    Figure 5.  Phenanthrene derivatives from Bletilla species (1−66)[3841,4345,47,49,58,7072,7479].
    Figure 6.  Phenanthrene derivatives from Bletilla species (67−105)[41,43,49,5961,70,76,7986].
    Figure 7.  Phenanthrene derivatives from Bletilla species (106−116)[43,49,70,75,84,85,87].

    Phenolic acids are carboxylic acids created from the skeletons of either benzoic or cinnamic acids[4648]. Fifty-eight phenolic acids (Figs 810, 117 to 174) were isolated from B. formosana, B. ochracea, and B. striata.

    Figure 8.  Phenolic acids from Bletilla species (117−134)[1,5,36,4752,54,67,88,90].
    Figure 9.  Phenolic acids from Bletilla species (135−169)[39,45,4854,56,61,68,69,73,76,82,83,8994].
    Figure 10.  Phenolic acids from Bletilla species (170−174)[20,95].

    For example, compounds 121, 126, 139, 141, 148, 149, 154, 155 and 157 were isolated from the rhizomes of B. formosana[1,49,5052]. The structures of these compounds were determined, mostly from their NMR spectroscopy data. Additionally, protocatechuic (136) and vanillin (137) also have been isolated from B. striata[53]. Moreover, some bioactive components such as 2-hydroxysuccinic acid (164) and palmitic acid (165) have been discovered and identified from B. striata[20,5456].

    The bibenzyls were small-molecular substances with a wide range of sources, which were steroidal ethane derivatives and resembling the structural moiety of bioactive iso-quinoline alkaloids[57].

    For example, depending on their structural characteristics, 70 bibenzyl compounds (Fig. 11, 175 to 244) can be grouped into three groups, simple bibenzyls (175186, 233238), complex bibenzyls (189225) and chiral bibenzyls (226-232, 239-244)[5860].

    Figure 11.  Bibenzyls from Bletilla species (175-244)[1,4042,47,49,50,5860,70,73,76,9699].

    Flavonoids are among the most common plant pigments. Eight bibenzyls (Fig. 12, 245 to 252) have been isolated from B. formosana, B. ochracea, and B. striata. Apigenin (245) and 8-C-p-hydoxybenzylkaempferol (249) were isolated from the whole plant of B. formosana[45]. Bletillanol A (250), bletillanol B (251) and tupichinol A (252) were isolated from B. striata[61]. The names and chemical structures of the flavonoids reported from Bletilla are shown below (Fig. 12).

    Figure 12.  Flavonoids from Bletilla species (245–252)[45,61].

    Twenty-four triterpenoids and steroids (Fig. 13, 253 to 276) have been reported from Bletilla (Fig. 13), such as, tetracyclic triterpenes (253259) and pentacyclic triterpenes (189225) and chiral bibenzyls (260)[6264]. Steroids (261276) isolated from the Bletilla and have shown some bioactivity. For example, bletilnoside A (272) was isolated from Bletilla species and displayed anti-tumor activity[65,66].

    Figure 13.  Triterpenoids and steroid compounds from Bletilla species (253-276)[56,6265].

    Thirteen other compounds (Fig. 14, 277 to 289) were isolated from B. formosana, B. ochracea, and B. striata. These compounds included amino acids, indoles and anthraquinones[67,68]. For example, syringaresinol (285) and pinoresinol (286) have been described in the methanol extract of the tubers of B. striata[61].

    Figure 14.  Others compounds from Bletilla species (277−289)[50,54,61,62,67,68,94,97].

    Based on the information about the chemical constituents of Bletilla species, it appears that there is a substantial body of research on these compounds. However, there are some areas that may warrant further investigation and research. At first, it would be valuable to investigate potential synergistic effects and interactions between the different classes of compounds within Bletilla species, as some of the compounds may work together. Besides, it is worth considering the improvement of compound yield. Optimizing extraction methods and finding the most efficient and environmentally friendly techniques are vital for both research purposes and potential commercial applications. It is also important to take into account the variability in chemical composition among different Bletilla species and even within the same species from different cultivars.

    The rich and varied chemical components make the plants of Bletilla have a wide variety of pharmacological activities (Table 4). Many studies have shown that the plants of this genus have anti-inflammatory, antineoplastic, antiviral, antioxidant, hemostatic, antibacterial, and other biological activities, which help to support the traditional medicinal practice of Bletilla in folk medicine.

    Table 4.  Summary of the pharmacological activities of Bletilla species.
    Pharmacological activityTested substance/partTested system/organ/cellTested dose/dosing methodResultsRefs.
    Anti-inflammatoryEthanol extract of Bletilla striataRAW264.7 cells RAW264.7 cells were pre-treated with ethanol extract of B. striata for 1 h and then stimulated with LPS (200 ng/mL) for 12 h, 0.05% DMSO was applied as the parallel solvent control. The culture supernatant was collected for IL-6 and TNF-α detection.Ethanol extract of B striata significantly inhibited LPS-induced interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression at 2.5 µg/mL.[41]
    The ethyl acetate-soluble (EtOAc) extract of tubers of B. striataH2O2-induced PC12 cell injury modelPC12 cells were seeded in 96-well multiplates at a density of 1.5 × 105 cells/mL. After overnight incubation at 37 °C with 5% CO2, 10 μM test samples and H2O2 (final concentration of 450 μM) were added into the wells and incubated for another 12 h.It protected the cells with the cell viabilities of 57.86 ± 2.08%, 64.82 ± 2.84%, and
    64.11 ± 2.52%.
    [98]
    Ethanol extract of tubers of B. striataRAW264.7 cellsCells were treated with ethanol extracts (25 μM) dissolved in DMSO, in the presence of
    1 μg/mL lipopolysacchride
    (LPS) for 18 h
    The anti-inflammatory activity with IC50 of 2.86 ± 0.17 μM.[54]
    PE extract of the tubers of B. striataLPS-stimulated BV2 cellsCells treated with extract
    (0, 1, 10, 30, 100 μg/mL) and dihydropinosylvi (0, 1, 10, 30, 100 μM) in presence of LPS
    (1 μg/mL)
    The anti-inflammatory activity with IC50 values of 96.0 μM.[96]
    Ethanol extract of the roots of B. striataCox-1 and Cox-2Treated with the ethanol extracts at various concentrations
    (0, 1, 10, 100 μM)
    The compounds with sugar moieties displayed selective inhibition of Cox-2 (N90%).[38]
    B. striata polysaccharide (BSPb)Human mesangial cells (HMCs)HMCs were pre-treated with BSPb (5, 10, 20 μg/mL)BSPb efficiently mediated expression of NOX4 and TLR2, to attenuate generation of ROS and inflammatory cytokines.[12]
    Compounds extracted from the rhizomes of Bletilla ochraceaRAW264.7 cellsAfter 24 h preincubation,
    cells were treated with serial dilutions in the presence of
    1 μg/mL LPS for 18 h. Each compound was dissolved in DMSO and further diluted in medium to produce different concentrations. NO production in the supernatant was assessed by adding 100 μL of Griess regents.
    It showed the inhibitory effects with IC50 values in the range of 15.29–24.02 μM.[76]
    Compounds extracted from the rhizomes of B. ochraceaMurine monocytic RAW264.7 cellsAfter 24 h preinubation, RAW 264.7 cells were treated with compounds (25 μM) dissolved in DMSO, in the prenence of
    1 μg/mL LPS for 18 h. NO production in each well was assessed by adding 100 μL of Giress regent
    It showed the inhibitory effects with IC50 2.86 ± 0.17 μM.[86]
    Compounds extracted from the rhizomes of Bletilla formosanaElastase Release AssaysNeutrophils (6 × 105 cells/mL) were equilibrated in MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM) at 37 °C for 2 min and then incubated with a test compound or an equal volume of vehicle (0.1% DMSO, negative control) for 5 min.Most of the isolated compounds were evaluated for their anti-inflammatory activities. The results showed that IC50 values for the inhibition of superoxide anion generation and elastase release ranged from 0.2 to 6.5 μM and 0.3 to 5.7 μM, respectively.[49]
    Anti-tumorTwo compounds from Bletilla striataA549 cellsCompounds were tested for their ability to induce ROS generation in A549 cells at concentrations of 20 two compounds for 24 h, the cells were harvested to evaluate the ROS production.The two compounds exhibited antiproliferative effects using the MTT test; these effects may be due to cell cycle arrest and inducing ROS generation.[87]
    Stilbenoids from B. striataBCRP-transduced K562 (K562/BCRP) cellsIt showed antimitotic activity and inhibited the polymerization of tubulin at IC50 10 μM.[78]
    Compounds extracted from the rhizomes of B. ochraceaThe human tumor cell lines HL-60 (acute leukemia), SMMC-7721 (hepatic cancer), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer)100 μL of adherent cells were seeded into each well of 96-well cell culture plates. After 12 h of incubation at 37 °C, the test compound was added. After incubated for 48 h, cells were subjected to the MTS assay.All isolated metabolites except 7 were evaluated for cytotoxic activity against five human cancer cell lines (HL-60, SMMC7721, A-549, MCF-7 and SW480).[76]
    AntiviralThe tuber of B. striataMadin-Darby canine kidney model and embryonated eggs modelAs simultaneous treatment with 50% inhibition concentration (IC50) ranging from 14.6 ± 2.4 to 43.3 ± 5.3 μM.Phenanthrenes from B. striata had strong anti-influenza viral activity in both embryonated eggs and MDCK models.[107]
    The 95% ethanol
    Extract of B. striata
    BALB/C miceIt has significant anti-influenza
    virus effect in mice, which may be related to the increase of IL-2, INFα, INF-β and thus enhance the immune function of mice.
    [12]
    AntioxidantCompounds extracted from the rhizomes of B. formosanaDPPH radical-scavenging assaySolutions containing 160 μL of various concentrations of sample extract, 160 μL of various concentrations of BHA, 160 μL of various concentrations of ascorbic acid, and the control (160 μL of 75% methanol) were mixed separately with 40 μL of 0.8 mM DPPH dissolved in 75% methanol. Each mixture was shaken vigorously and left to stand for 30 min at room temperature in the dark.Tthe seedlings grown by tissue culture of B. formosan collected in Yilan County had the best antioxidant capacity. In addition, B. formosana collected in Taitung County had the best scavenging capacity in the tubers, leaves and roots.[93]
    Fibrous roots of B. striataDPPH model and superoxide anion systemThe ABTS+ solution was prepared by reacting 7 Mm ABTS with 2.45 mM potassium persulfate (final concentrations both dissolved in phosphate buffer, 0.2 M, pH 7.4) at room temperature for 12–16 h in the dark.It removed free radicals and inhibit tyrosinase activity.[33]
    B. striata extracts (BM60)The murine macrophage cells NR8383, male SD mice (180~200 g)NR8383 were pretreated with extracts (1, 10 and 100 g/mL) for 4 h and then 65 stimulated with 1 g/mL of LPS for 24 h. Acute lung injury was induced in mice by nonhexposure intratracheal instillation of LPS (3.0 mg/kg). Administration of the BM60 extract of 35, 70, and 140 mg/kg (L, M, H) was performed by oral gavages.The BM60 treatment reduced the production of NO in NR8383 macrophages. Treatments with BM60 at the doses of 35, 70, 140 mg/kg significantly reduced macrophages and
    neutrophils in the bronchoalveolar lavage fluid (BALF).
    [12]
    The crude
    polysaccharides obtained from B. striata
    DPPH free radical scavenging activityConcentration
    range of 2.5–5.0 mg/mL
    The IC50 of BSPs-H was 6.532 mg/mL.[35]
    HemostasisB. striata polysaccharide (BSP)Diabetes mellitus (DM) mouse models were induced by high fat-diet feeding combined with low-dose streptozocin injectionDM mouse models were induced by high fat-diet feeding combined with low-dose streptozocin injection. The BSP solutions were applied on the surface of each wound at a volume of 50 μl. RD mice were assigned as normal controls and received saline treatment (n = 6). All mice were treated with vehicle or BSP once daily from the day of wounding (d0) until 12 days later (d12).BSP administration accelerated diabetic wound healing, suppressed macrophage infiltration, promoted angiogenesis, suppressed NLRP3 inflammasome activation, decreased IL-1β secretion, and improved insulin sensitivity in wound tissues in DM mice.[112]
    B. striata Micron Particles (BSMPs)Tail amputation model and healthy male Sprague-Dawley (SD) rats
    (250 ± 20 g, 7 weeks of
    age)
    Rats were divided into six groups of five treated with cotton gauze and BSMPs (350–250, 250–180, 180–125, 125–75, and < 75 μm), respectively.Compared to other BSMPs of different size ranges, BSMPs of 350–250 μm are most efficient in hemostasis. As powder sizes decrease, the degree of aggregation between particles and hemostatic BSMP effects declines.[109]
    Rhizoma Bletillae polysaccharide (RBp)Adult male SD rats weighing 220 ± 20 gAfter incubation for 1 min at 37 °C, 300 μL of PRP was dealt with different concentrations of RBp (50, 100, 150, and 200 mg/L) under continuous stirring, and the vehicle was used as the blank control.RBp significantly enhanced the platelet aggregations at concentrations of 50−200 mg/L in a concentration-dependent manner.[113]
    AntibacterialBibenzyl derivatives from the tubers of Bletilla striataS. aureus ATCC 43300, Bacillus subtilis ATCC 6051, S. aureus ATCC 6538 and Escherichia coli ATCC 11775Using a microbroth dilution method, bacteria were seeded at
    1 × 106 cells per well (200 μL) in a
    96-well plate containing Mueller-Hinton broth with different concentrations (from 1 to 420 μg/mL, 300 μg/mL and so on;
    2-fold increments) of each test compound.
    It showed inhibitory activities with MIC of (3–28 μg/mL) against S. aureus ATCC6538[116]
    The crude extract of B. striataS. album, A. capillaris, C. cassiaThey were seeded at 1 × 106 cells per well (200 μL) in a 96-well plate containing Mueller−Hinton broth (meat extracts 0.2%, acid digest of casein 1.75%, starch 0.15%) with different concentrations (from 1 to 128 μg/mL; 2-fold increments) of each test compound.It showed S. album (0.10%), A. capillaris (0.10%), and C. cassia (0.10%) to have the strongest antibacterial properties.[118]
    The ethyl acetate-soluble (EtOAc) extract of tubers of B. striataS. aureus ATCC 43300, S. aureus ATCC 6538, and Bacillus subtilis ATCC 6051) and Escherichia coli ATCC 11775)Bacteria were seeded at 1 × 106 cells per well (200 μL) in a 96-well plate containing Mueller Hinton broth with different concentrations (from 1 to 420 μg/ml; 2-fold increments) of each test compound.The extract was effective against three Gram-positive bacteria with minimum inhibitory concentrations (MICs) of 52–105 μg/ml.[98]
    The phenanthrene fraction (EF60) from the ethanol extract of fibrous roots of Bletilla striata pseudobulbsS. aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 43300, E. coli ATCC 35218, and P. aeruginosa ATCC 27853, Bacillus subtilis 168EF60 was active against all tested strains of Staphylococcus aureus, including clinical isolates and methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentration (MIC) values of EF60 against these pathogens ranged from 8 to 64 μg/mL.EF60 could completely kill S. aureus ATCC 29213 at 2× the MIC within 3 h but could kill less than two logarithmic units of ATCC 43300, even at 4× the MIC within 24 h. The postantibiotic effects (PAE) of EF60 (4× MIC) against strains 29213 and 43300 were 2.0 and 0.38 h, respectively.[117]
    Anti-adhesiveBletilla striata extraction solutionPPA was induced by cecal wall abrasion, and Bletilla striata was injected to observe its efect on adhesion in ratsThe rats in the sham operation group was not treated; the other rats of the three experimental groups were intraperitoneally injected with 8 ml of phosphate-buffered saline (Control group), 15% Bletilla striata extraction solution (BS group), and 0.2% hyaluronic acid solution (HA group), respectively.Bletilla striata decreased the development of abdominal adhesion in abrasion-induced model of rats and reduced the expression of the important substance which increased in PPAs.[120]
    ImmunomodulatoryB. striata polysaccharide (BSPF2)Mouse spleen cellsTo observe the immune activity of BSPF2, mouse spleen cells were stimulated with BSPF2 at 10–100 g/mL for 72 h.Immunological assay results demonstrated that BSPF2 significantly induced the spleen cell proliferation in a dose-dependent manner.[121]
    Anti-pulmonary fibrosisB. striata polysaccharideClean grade male SD ratsSD rats were randomly divided into 5 groups, sham operation group (equal volume of normal saline), model group (equal volume of normal saline), tetrandrine positive control (24 mg/kg) group and white and Polysaccharide low
    (100 mg/kg) and high (400 mg/kg) dose groups.
    The Bletilla striata polysaccharide has remarkable regulation effect on anti-oxidation system and immune system, but cannot effectively prevent lung fibrosis.[127]
    Small molecule components of Bletilla striataClean grade male SD ratsSD rats were randomly divided into 5 groups, sham operation group (0.5 mL normal saline), model group (0.5 mL normal saline), and positive control group (tetrandrine 24 mg/kg) and low (20 mg/kg) and high (40 mg/kg) dosage groups of the small molecule pharmacological components of Bletilla, which were administered by gavage once a day for 2 consecutive months.The small molecule components of Bletilla striata can effectively prevent lung fibrosis though regulating the anti-oxidation system,immune system and cytokine level; SMCBS is one of the active components of Bletilla striata on silicosis therapy[124]
    —, not given.
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    Many phytochemicals have been well characterized to lessen swelling or inflammation[89]. A series of phenolic acid and polysaccharide compounds isolated from Bletilla demonstrated anti-inflammatory bioactivity against BV-2 microglial, RAW 264.7, and PC12 cells[96,100102]. For example, phochinenin K (106) exhibited growth inhibitory effects with an IC50 of 1.9 μM, and it is a possible candidate for development as neuroinflammation inhibitory agent[43]. Using the H2O2-induced PC12 cell injury model, (7S)-bletstrin E (242), (7R)-bletstrin F (243) and (7S)-bletstrin F (244) could clearly protect the cells with the cell viabilities of 57.86% ± 2.08%, 64.82% ± 2.84%, and 64.11% ± 2.52%, respectively[98]. With an IC50 of 2.86 ± 0.17 μM, 2,7-dihydroxy-4-methoxyphenanthrene (53) showed potential action against NO generation in RAW 264.7 macrophages[54]. The use of Bletilla in traditional skin care, it is said to function as an astringent, hemostatic and wound healing[33]. Modern medical pharmacology research has validated that this plant has antibacterial effects, which may may help to explain, in part, its traditional use in skin care[24].

    Though it's mentioned that some of these compounds from Bletilla have demonstrated anti-inflammatory action, more extensive studies are needed to fully understand their mechanisms of action, potential therapeutic applications, and safety profiles. Conducting in vivo studies and clinical trials can provide more concrete evidence of their effectiveness.

    There are important antineoplastic agents that have originated from plant natural products[103]. In recent years, several bibenzyl and flavonoid compounds have been discovered from Bletilla that have antineoplastic activity against A549 cells and other cells. For example, 7-hydroxy-2-methoxy-phenanthrene-3,4-dione (160) and 3′,7′,7-trihydroxy-2,2′,4′-trimethoxy-[1,8′-biphenanthrene]-3,4-dione (163) have shown strong antiproliferative effects and induced ROS production after 24 h in A549 cells[87]. The doxorubicin (Dox)/FA (folate)-BSP-SA (stearic acid) modified Bletilla striata polysaccharide micelles boosted the drug enrichment in tumors and improved the in vivo anticancer effects[104,105]. Micelles, nanoparticles, microspheres, and microneedles are examples of B. striata polysaccharide-based drug delivery systems that exhibit both drug delivery and anti-cancer functionality. These experiments confirmed that some of the compounds isolated from the Bletilla have potential activity for the treatment of cancer.

    However, most of the evidence presented in the previous studies is based on in vitro experiments or cell culture studies. It is highly necessary to use animal models to study the in vivo anti-tumor effects of Bletilla extracts or compounds. These studies can help evaluate the safety and effectiveness of treatments based on Bletilla. Additionally, through such methods, researchers can further investigate the mechanisms of Bletilla's anti-tumor activities, exploring how Bletilla compounds interact with cancer cells, immune responses, and signaling pathways involved in tumor growth and metastasis.

    Antiviral medications are essential for preventing the spread of illness, and are especially important nowadays with pandemics and drug-resistant viral strains[5, 6]. Therefore, it is vitally necessary to find novel, safe, and effective antiviral medications to treat or prevent viral infections[106]. B. striata plant contains compounds that have been recorded in ancient texts to cure cough, pneumonia, and skin rashes, and these may be related to potential antiviral constituents[23]. Some constituents of B. striata have antiviral activity, for example, phenanthrenes and diphenanthrenes from B. striata displayed potent anti-influenza viral in a Madin-Darby canine kidney model and embryonated eggs model, diphenanthrenes with parentally higher inhibitory activity than monophenanthrenes[107]. But more research is needed to further determine the antiviral activity of Bletilla, understand how Bletilla compounds interact with viral proteins or the host immune response, and conduct safety and toxicity studies, which are crucial for the development of related materials.

    Free radicals have the potential to exacerbate lipid peroxidation and harm cell membranes, which can lead to several prevalent human diseases, including cancer, cataracts, and coronary heart disease[108]. Research has shown that extracts from Bletilla possess strong antioxidant activity. However, this antioxidant activity can vary depending on the different growing environments of the plant. Additionally, the antioxidant capabilities of extracts from different parts of the Bletilla plant also vary[93]. Clinical studies have shown that traditional Chinese medicine formulas containing Bletilla can inhibit tyrosinase activity and possess antioxidant properties, thus resulting in skin-whitening effects[108]. Furthermore, some research reveals that the polysaccharides in the plant exhibit significant antioxidant activity, effectively scavenging free radicals and inhibiting tyrosinase activity[33]. This highlights the skin-whitening potential of the fibrous root of Bletilla striata, indicating promising prospects for the comprehensive utilization of the B. striata plant[33]. However, most studies on the pharmacological activities of Bletilla have focused solely on B. striata, neglecting other species within the genus. Different species may possess varying phytochemical compositions and antioxidant properties, which can lead to an incomplete understanding of the genus as a whole.

    Available hemostatic agents are expensive or raise safety concerns, and B. striata may serve as an inexpensive, natural, and promising alternative[109]. Polysaccharides of B. striata displayed hemostatic activity through inhibition of the NLRP3 inflammasome[110112]. The ADP receptor signaling pathways of P2Y1, P2Y12, and PKC receptors may be activated as part of the hemostasis[113]. Alkaloids from Bletilla have hemostatic activities through platelet deformation, aggregation, and secretion. In addition, polysaccharides of Bletilla striata have potential wound-healing medicinal value[110]. Currently, Bletilla plants have been used in various traditional systems, such as traditional Chinese medicine and Ayurveda, to control bleeding.

    Previous studies revealed that Bletilla displayed antibacterial effects[114]. For example, bletistrin F, showed inhibitory activities with MIC of (3–28 μg/mL) against S. aureus ATCC 6538[115,116]. Antimicrobial screening of Bletilla showed S. album (0.10%), A. capillaris (0.10%), and C. cassia (0.10%) to have the strongest antibacterial properties[117,118]. In addition, phenanthrenes are worthy of further investigation as a potential phytotherapeutic agent for treating infections caused by S. aureus and MRSA[119]. However, further in vivo studies on the antibacterial activity of Bletilla are lacking, which is needed for clinical application. For example, the specific mechanism of antibacterial activity of Bletilla still needs to be elucidated. While research on the antibacterial activity of Bletilla plants is promising, it faces several shortcomings and challenges that need to be addressed for a more comprehensive understanding of their potential therapeutic applications. Further studies with standardized methodologies, mechanistic insights, clinical trials, and consideration of ecological and safety concerns are essential to advance this field.

    There are other pharmacological activities of Bletilla, like anti-fibrosis activity, anti-adhesive activity, and immunomodulatory activity. For example, B. striata has been studied as a new and cheaper antiadhesive substance which decreased the development of abdominal adhesion abrasion-induced model in rats[120]. However, the natural resources of Bletilla are also getting scarcer. To preserve the sustainable development of Bletilla species, proper farming practices are required, along with the protection and economical use of these resources. The immunomodulatory activity of the Bletilla species was assessed using the 3H-thymidine incorporation method test, and BSP-2 increased the pinocytic capacity and NO generation, which improved the immunomodulatory function[121,122].

    B. striata extract was shown to have anti-pulmonary fibrosis effect[123]. B. striata polysaccharide can successfully prevent lung fibrosis through established by invasive intratracheal instillation method and evaluated by lung indexes[123,124]. Moreover, Bletilla species need further investigations to evaluate their long-term in vivo and in vitro activity before proceeding to the development of pharmaceutical formulation.

    While there is currently a deep understanding of the pharmacological activity of plants in the Bletilla genus, there are still many gaps that need to be addressed. To overcome these shortcomings, future research on the pharmacological activity of Bletilla species should emphasize comprehensive, well-designed studies with a focus on species-specific effects, mechanistic insights, and rigorous clinical trials. Additionally, collaboration among researchers, standardization of methods, and transparent reporting of results can help advance our understanding of the therapeutic potential of Bletilla plants. Researchers should also consider safety aspects and explore potential herb-drug interactions to ensure the responsible use of Bletilla-based therapies.

    There are several common clinical applications of Bletilla striata in TCM. The gum of B. striata has unique viscosity characteristics and can be used as thickener, lubricant, emulsifier and moisturizer in the petroleum, food, medicine, and cosmetics industries[125130]. B. striata is used as a coupling agent, plasma substitute, preparation adjuvant, food preservative and daily chemical raw material[131133]. In clinical practice, B. striata glue has also been proven to control the infections and is beneficial to the healing of burns and wounds[133135].

    In ethnic communities in Southwest China, the locals chew fresh Bletilla tubers directly or take them orally after soaking in honey to treat cough, pneumonia and other diseases[33, 34]. This traditional use is common in local communities in Southwest China, and suggests at the safety of Bletilla. However, current research shows it is still necessary to control the dosage when using Bletilla[136].

    Zebrafish embryos and larvae respond to most drugs in a manner similar to humans[137]. Militarine, the main active ingredient of Bletilla, was tested in a zebrafish embryo development assay at concentrations of 0.025 g/L and 0.05 g/L, and with the increased concentration, the heart rate of zebrafish embryos is slowed. Mortality and malformation rates of zebrafish embryos gradually increased with time and militarine concentration[138]. Although Bletilla species are safe at therapeutic dose ranges, further research on their safety is required[136]. More in-depth studies should be carried out on Bletilla to extract effective ingredients and make better preparations for clinical use[139].

    According to the traditional medicinal knowledge in ancient Chinese texts, Bletilla has been an important ingredient for skin care since ancient times. Many ethnic minority groups in China still retain the practice of using Bletilla for skin care, and the plant parts and preparation methods of use are consistent with the records in ancient texts. Almost 300 phytochemicals have been identified from Bletilla, and some of them possess important pharmacological activities, which support its traditional uses and suggest the important medicinal development potential of this genus. This review has demonstrated that Bletilla, as an important medicinal plant of Orchidaceae, still requires further research to fathom its medicinal potential.

    For instance, it is necessary to enhance the quality control procedures based on the chemical components and pharmacological activity of Bletilla. The chemical composition and pharmacological properties of Bletilla are critical areas of current research. According to previous studies, the main bioactive components of Bletilla can vary greatly according to its origin, harvest time, distribution, storage, and adulteration. However, variation in bioactivities caused by the differences in Bletilla constituentshave not been explored extensively yet. To develop clinical applications of Bletilla, it is crucial to further explore the mechanism of action between its chemical composition variation and its pharmacological actions.

    In addition, although the tuber has historically been the main medicinal part of Bletilla, research has shown that the chemical composition in other parts of Bletilla, such as stems, leaves, and flowers, also give these parts a variety of pharmacological activities. Further in-depth analysis of the chemical components and pharmacological activities of different parts of this genus is worthwhile, to explore the specific chemical basis of its pharmacological activities, develop related drugs, and promote clinical applications. For example, Bletilla polysaccharide has good hemostasis and astringent wound effects[110], so it may have the potential to be developed into a drug or related medical materials to stop bleeding and heal wounds.

    Finally, as a cautionary note, many unrestrained collections and the destruction of habitats have made the resources of wild Bletilla rarer. In addition to protecting the wild populations of Bletilla, appropriate breeding techniques should be adopted to meet the commercial needs of this economically important genus, thereby allowing its sustainable use in commerce.

    The authors confirm contribution to the paper as follows: study conception and design, funding acquirement: Long C; data analysis, draft manuscript preparation, literature review: Fan Y, Zhao J; manuscript revise and language editing: Wang M, Kennelly EJ, Long C. All authors reviewed the results and approved the final version of the manuscript.

    The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation, to any qualified researcher. Requests to access these datasets should be directed to Yanxiao Fan (fanyanxiao0510@163.com).

    This research was funded by the Yunnan Provincial Science and Technology Talent and Platform Plan (202305AF150121), Assessment of Edible & Medicinal Plant Diversity and Associated Traditional Knowledge in Gaoligong Mountains (GBP-2022-01), the National Natural Science Foundation of China (32370407, 31761143001 & 31870316), China Scholarship Council (202206390021), and the Minzu University of China (2020MDJC03, 2022ZDPY10 & 2023GJAQ09).

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

  • Supplemental Table S1 Functional validation information of Flower color.
    Supplemental Table S2 Functional validation information of Flower morphology.
    Supplemental Table S3 Functional validation information of Weeping traits.
    Supplemental Table S4 Functional validation information of other resistance.
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  • Cite this article

    Fan D, Miao R, Lv W, Wen Z, Meng J, et al. 2024. Prunus mume genome research: current status and prospects. Ornamental Plant Research 4: e006 doi: 10.48130/opr-0024-0004
    Fan D, Miao R, Lv W, Wen Z, Meng J, et al. 2024. Prunus mume genome research: current status and prospects. Ornamental Plant Research 4: e006 doi: 10.48130/opr-0024-0004

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Prunus mume genome research: current status and prospects

Ornamental Plant Research  4 Article number: e006  (2024)  |  Cite this article

Abstract: Mei (Prunus mume) is an excellent garden tree highly praised in China, possessing both ornamental and cultural values. Breeding Mei with distinctive characteristics and high resistance has become a long-term goal to meet the visual and spiritual demands in the new era. With the rapid development of biotechnology, researchers have successively completed the whole genome sequence and resequencing of Mei, and continue to employ advanced techniques to investigate the formation mechanisms of important ornamental traits and stress resistance traits in Mei. Thus, the groundwork has been established for achieving the breeding objectives. In this article, we provide an overview of the development and expansion of genome projects over the past decade, including whole-genome sequencing, resequencing, and genetic mapping. We further present a concise summary of the research progress made in understanding major ornamental traits and cold resistance traits. These accomplishments hold great promise for significantly enhancing the efficiency of Mei and further realizing breeding goals.

    • Genomics is a scientific field that encompasses genome mapping, sequencing, and functional analysis of an organism's entire genome. It aims to unravel the complete genetic information encoded in the genome. Plant genomes, in particular, pose significant challenges due to enlarged sizes, complexity resulting from transposable elements, and a long history of genome duplications. The establishment of the Arabidopsis genome sequence in 2,000 has played a vital role in advancing plant genomics and established Arabidopsis as a popular species for fundamental plant research[1]. In recent years, significant advancements have been made in the field of plant genomics, including the development of innovative sequencing technologies and bioinformatic tools, enabling faster, more efficient, and cost-effective genome sequencing and assembly. Currently, approximately 400 plant genomes have been sequenced. This progress has provided a wealth of genetic data for studying plant diversity and has empowered breeders to conduct extensive multidimensional studies in the domains of genetics, genomics, and molecular breeding. These advancements have opened up novel prospects and catalysts for the breeding of various plant species, leading to a revolution in breeding technology.

      Prunus mume, also known as Mei, is a classic and renowned flower indigenous to China, ranking among the top ten most famous flowers in the country. It has been cultivated for ornamental purposes and as an important fruit tree for over 3,000 years. The use of P. mume dates back even further, with evidence of their cultivation during the Neolithic period, around 5,000−7,000 years ago[2,3]. The cultivation and evolution of modern Mei varieties have progressed through various stages, including wild Mei, fruiting Mei, flowering Mei, and both flowering and fruiting Mei[4]. Initially, Chinese ancestors began cultivating wild Mei primarily for its edible and medicinal properties. Over time, they also recognized its ornamental value and expanded its cultivation for aesthetic purposes. China plays a crucial role in both the origination and cultivation of plum blossoms, demonstrating its significance as the hub for their genetic diversity and variations. Throughout its long history, Mei populations have undergone continuous evolution and selective breeding[5]. As a result, the vibrant and diverse Mei varieties that we observe today have emerged. These varieties showcase a wide range of colors and characteristics, representing the culmination of centuries of cultivation and selection efforts.

      In less than ten years, more than 65 ornamental plants have had their full genomes sequenced, after the completion of the genome sequencing of the first ornamental plant in 2012[6]. The completion of the P. mume genome sequencing has provided a solid platform for other ornamental plants to be studied so that chromosome evolution, genome structure and patterns of genetic variation can be described. A high-quality P. mume genome sequencing has made it possible to identify a number of gene families that regulate desirable and profitable features. Additionally, significant progress has been made in the establishment of fresh genetic mapping projects, the investigation of genome evolution, and the creation of sturdy and dependable molecular markers[7]. The efforts to improve genetic makeup will continue to be greatly impacted by this new information and the resources that are now available.

    • Since 1998, the Mei research team at Beijing Forestry University (China) has been dedicated to studying the mechanisms behind the production of significant traits[8]. By utilizing methods from molecular biology, their research aims to enhance breeding efficiency and establish a solid theoretical foundation for molecular design breeding in Mei. Future targeted and effective breeding techniques would be made possible by the work, which has deepened our understanding of the genetic foundation of desirable traits[8]. The genetic background of Mei has been a challenging aspect to understand given its complicated gene control network, lengthy breeding cycle, and ambiguous genetic composition, and difficulties in studying molecular mechanisms related to traits such as flower fragrance, cold resistance, flower type, and flower bud development, etc. Carrying out whole genomics research is an effective means to break through the research bottleneck. The P. mume Genome Project, launched in 2009, is a collaboration between the National Research Center for Flower Growing Engineering at the Beijing Genome Institute (BGI) and Beijing Forestry University. The wild Mei from Tongmai in Tibet was collected for whole-genome sequencing using the Illumina Genome Analyzer (GA) II method. The wild Mei in this region is highly homozygous due to its closed geographical environment. The final 28.4 Gb clean data was generated by correcting and filtering with 94.7× sequencing depth[6]. Whole genome mapping (WGM) was applied to improve the assembly of the genome. Repetitive sequences, including tandem repeated sequence and interspersed repeated sequence, were extracted from the P. mume genome, with TEs making up 97.9% of these repetitive sequences. The Copia and Gypsy long terminal repeat families were the most abundant TEs in the P. mume genome, aligning with the apple (Malus domestica) genome. The P. mume genome contains 287 small nuclear RNAs, 125 ribosomal RNAs, 209 microRNAs, and 508 transfer RNAs. The Rosaceae family, comprising over 100 genera and 3,000 species, includes fruits, nuts, and ornamental plants with medicinal and ornamental values. Whole-genome sequencing in apple[9], strawberry[10] and Mei[6] , are the foundation for the construction of ancestral chromosomes of Rosaceae and the study of chromosome evolution among Rosaceae. Nine ancestral chromosomes were constructed, with seven strawberry chromosomes produced after 15 fusions and 17 apple chromosomes after one genome replication and five fusions. Eight current chromosomes were created by 11 fusions, whereas its chromosomes 4, 5, and 7 were directly derived from the ancient chromosomes III, VI, and VI, respectively, without undergoing any rearrangement. Special floral scent and early blooming are important properties of Mei. The formation of the distinctive scent of Mei may be explained by the identification of benzyl alcohol acetyltransferase (BEAT) genes that control the production of phenylmethyl acetate, the primary aroma component[6,11]. Six dormancy-associated MADS box (DAM) genes related to dormancy were found to be distributed in tandem and repeated, and six C-repeat binding factors (CBF) gene binding sites were found to be upstream of the DAM gene[6]. Previous research[12] has indicated that the DAM gene and multiple CBF binding sites are significant factors in the early release of dormancy in Mei, making them very sensitive to temperature changes that lead to a short dormancy period and early blooming. These findings are relevant to the investigation of flowering related genes and the molecular mechanism of breaking bud dormancy at low temperature may account for early spring blossoming. Moreover, pathogenesis-related (PR) proteins, which are proteins encoded by plants in response to a variety of stressors, were also identified in P. mume genome. PR10 gene families were significantly and highly expressed in its roots and leaves, as a result, the expansion of the PR10 family may be connected to the plant's reaction to fungus, salinity, and drought in its roots and leaves[13,14]. On the other hand, P. mume genomes were de novo assembled in recent studies, yielding assembly sizes of 241.72 Mb and contig N50 of 3.35 Mb. 31,116 gene models in total were annotated[15]. Recently, the genome sequencing and de novo assembly of P. mume var. tortuosa were performed successfully utilizing Oxford Nanopore technology (ONT). Produced a 237.8 Mb genome assembly that has an anchoring rate of 98.85 when anchored onto eight pseudo chromosomes. In contrast to an earlier draft genome from wild P. mume that had a lower scaffold N50 value (577.82 kb) and contig N50 value (31.77 kb). The recently assembled genome demonstrates substantial enhancements, with a scaffold N50 of 29.4 Mb and a contig N50 of 2.75 Mb[16]. The genome sequencing of Mei and its variants P. mume var. tortuosa has provided a solid framework for exploring the mechanisms that aid in the formation of various essential traits in Mei. The chloroplast genome, as one of the three major genomes in plant cells, is not only small in size compared to the nuclear genome, but it also has relatively independent genetic material chloroplast DNA and a highly conserved genome structure. As a result, the chloroplast genome is frequently regarded as an ideal system for phylogenetic research. In the study of chloroplast genome of Mei, the nuclear and chloroplast genomes of 19 fruit Mei varieties were sequenced[17], and the genetic diversity of fruiting Mei 0.096−0.134 was higher than that of ornamental Mei 2.01 × 10−3. The results showed that natural selection was more advantageous in the domestication process, while ornamental Mei experienced more artificial domestication to meet the needs, resulting in low genetic diversity[17,18]. Besides, mitochondrial genome sequencing can produce more useful SSR molecular markers for the study of species diversity. Currently, the apple[19], strawberry[20], and other species that are similar to Mei were the only ones with pertinent studies on the mitochondrial genomes of Rosaceae; the systematic studies have not been reported in Mei.

    • The purpose of whole genome resequencing (WGR) is to examine genetic variation in Mei with known genome sequences. Sequence alignment allows for the discovery of numerous single nucleotide polymorphisms (SNP), insertion/deletions (InDels), structural variants (SVs), and copy number variations (CNV). These genetic loci information can lay the foundation for population genetics, genome wide association analysis (GWAS) and pan-genome studies. A total of 15 wide individuals and 333 cultivars of Mei as in addition to its most closely related relatives, including Prunus davidiana, Prunus salicina and Prunus sibirica were sampled and sequenced for whole genome sequencing from Wuhan of Hubei Province, Qingdao of Shandong Province, Sichuan Province, Kunming of Yunnan Province, Lijiang of Yunnan Province and Guizhou Province[18]. The Chinese Mei classification system allows for the division of 333 Mei cultivars into 11 cultivar groups, which include Pendulous, Single Flowered, Versicolor, Pink Double, Flavescens, Tortuosa, Green Calyx, Alboplena, Cinnabar Purple, Apricot Mei, and Meiren[4]. After WGR analysis, a total of 1,298,196 SNPs were explored, including 733,292 non-synonymous, 7,313 deletion, 1,117 insertion and 623 structural variants[17]. Utilizing all of the high-quality SNPs observed, phylogenetic relationships among those 351 accessions were created, making use of three more Prunus species for the outgroup. With high confidence, 16 subgroups could be formed from the 348 Mei accessions, with 91.1% of the nodes (318/349) having a bootstrap value greater than 90. Pink Double and Single Flowered exhibit lower linkage disequilibrium (LD) than the natural population, according to linkage disequilibrium analysis results. This is likely due to large introgressions of other species into both of those subpopulations. Mei was divided into True Mei, Apricot Mei and Meiren in accordance with a previous study[2]. The introgression events were analyzed using the three-population F3 test, which showed significant inter-species introgression within the Mei and Prunus species. This resulted in a complicated population structure and analysis of the history of domestication. Genomes of nine Mei and four closely related species like P. sibirica, P. davidiana, P. salicina, and P. persica were sequenced to build a pan-genome. Core genes, 19,135 and 22,499 were sequenced in Mei and Prunus, respectively. There were 3,364 Mei-specific genes in the P. mume genome that were relatively enriched such as flavonoid, phenylpropanoid, stilbenoid, diarylheptanoid and gingerol biosynthesis, along with phenylalanine metabolism, potentially influencing ornamental traits like xylem color, flower color, and floral scent[18]. Evolutionary history of Prunus genus was reconstructed using 13 distinct Prunus accessions with three sequenced closely related species in Rosaceae, and the results showed that P. sibirica may be closer related to Mei than any other Prunus species[18]. It was estimated that 3.8 million years ago (MYA) there was an extinction gap among Mei with other Prunus species, and a 2.2 MYA extinction gap among wild and cultivated Mei. These divergence times significantly precede the projected domestication of cultivated Mei. Furthermore, 129 genomes of Prunus plants, including peach, apricot, and plum, underwent resequencing. By examining the genomes of 79 resequencing Mei varieties[18], the interspecific connectivity of Mei and related species was further ascertained. Numerous naturally occurring and purposely selected locations from interspecific infiltration were discovered, and they had a significant role in the current Mei population's formation[21]. A GWAS method based on logistic regression for 24 Mei traits was established. At the same time, RNA-Seq analysis was done on two typical cultivars 'Wuyuyu' (double red petals, purplish red sepals) and 'Mi Dan Lv' (single white petals, green sepals) was also performed. Based on the examination about the two sets of data mentioned above, the presence of 76 SNPs from different expression genes (DEGs) were found on chromosome Pa4 from 229 kb to 5.57 Mb, which had been linked to petal, stigma, calyx and bud color, accordingly, such as MYB108 (Pm012912, Pa4: 411731-413009), encoding an R2R3 MYB transcription factor, was associated with the anthocyanin metabolism pathway. Moreover, using combined GWAS with quantitative trait locus (QTL) mapping[22] , two regions linked with xylem color and filament color on chromosome 3 were localized, including R1 (20601577) controlling xylem color and R2 (444623-3375607) controlling filament color.

    • A genetic linkage mapping depicts the distribution of recombination through a genome from assigning genetic markers within linkage groups and ranking and positioning those markers based on recombination patterns among them. Given their abundance, stability, codominance, efficiency, and ease of automation, single nucleotide polymorphisms (SNPs), simple sequence repeats (SSRs), amplified fragment length polymorphism (AFLPs), random amplification of polymorphic DNA (RAPD) are important molecular markers which have been widely used for establishing high-density genetic maps. Zhang constructed a F1 generation with multi-petal characters of P. mume and discussed the feasibility of using the 'pseudo-test cross' method to construct a molecular genetic map[22]. Thus, the first genetic map of Mei was produced. Subsequently, based on the development of SSRs, Huang et al. built the framework genetic map by AFLPs and SSRs of Mei using 56 F1 generation of 'Xuemei' × 'Fengpi Gongfen'[23]. A 668.7 cM-long genetic linkage map was constructed with F1 individuals 'Fenban' × 'Kouzi Yudie', by 144 SSR markers. Seventy-one scaffolds comprising approximately 28.1% of the entire assembled P. mume genome were anchored to the genetic map[7]. Soon after, the restriction-site associated DNA sequencing (RAD-seq) method was then used to identify hundreds of thousands of SNPs for 'Fenban' and 'Kouzi Yudie' relying on the Mei reference genome. F1 family of 'Fenban' × 'Kouzi Yudie' was genotyped for SNPs[24]. By adding the selected 1,484 SNPs with the SSR linkage map, a high-density genetic map for P. mume containing a total length of 780.9 cM and eight linkage groups was created. A total of 513 scaffolds with a size of 199 Mb were attached to the genetic map, covering 84.0% of the assembled P. mume genome[24]. Additionally, 84 QTLs affecting stem growth and form, leaf morphology, and leaf anatomy were detected, among which the maximum number of QTLs controlling leaf area and vein number was 35, and the minimum number of QTLs controlling stem diameter was one[25]. The functional mapping framework ('Fenban' 'Kouzi Yudie', 'Liuban' 'Sanlun Yudie', and 'Liuban' 'Huang Lve') incorporates developmental allometry equations to map particular QTLs controlling the development of various phenotypes. These QTLs were integrated into a complex network using evolutionary game theory, and 'pioneering' QTLs (piQTLs) and 'maintaining' QTLs (miQTLs) were detected, which control how shoot height varies with diameter and how shoot diameter varies with height, respectively[26]. Subsequently, another study found that a small region of chromosome 1 (5−15 Mb) has a lot of floral QTLs[27]. Up to now, using 387 individuals developed from Mei cultivar 'Liuban' × 'Fentai Chuizhi'. This genetic linkage map has eight linked groups, including 8,007 genetic markers and the mean marker distance of 0.195 cM. The map's entire length was 1,550.62 cM, or 64.31% of genome. The F1 population yielded 66 QTLs linking 15 plant architecture and significant features connected to flowers. Using the P. mume genome's annotation information, 58 potential candidate genes were examined. Subsequently, the weeping phenotype in Mei was successfully mapped to the genomic regions spanning from 10.54 Mb to 11.68 Mb on chr7. Through this investigation, 10 specific SLAF molecular markers were found to be strongly linked to the weeping trait. Further investigation revealed that nine potential genes were significantly linked to the formation and development of the cell wall, as well as the cellulose synthesis and degradation. Additionally, another set of nine genes predicted to be involved in transcriptional regulation were speculated to play an essential part in the development of the weeping traits observed in Mei[28].

    • The completion of whole-genome sequencing, resequencing, and the construction of a high-density genetic map for Mei has established a crucial basis for analyzing the genetic regulatory mechanisms underlying important ornamental traits and facilitating molecular marker-assisted breeding. As a result, significant advancements have been made in understanding the genetic mechanisms governing flower scent, color, morphology, weeping traits, and resilience against abiotic stresses in Mei (Fig. 1).

      Figure 1. 

      Eleven cultivar groups of Mei and representative functional genes in five aspects. 1, Versicolor Group; 2, Dragon Group; 3, Pendent Group; 4, Cinnabar Purple Group; 5, Single Flowered Group; 6, Flavescens Group; 7, Blireiana Group; 8, Apricot Mei Group; 9, Pink Double Group; 10, Alboplena Group; 11, Green Calyx Group.

    • Flower scent is a highly valued quality trait in ornamental plants. In the natural environment, numerous plant species release floral scents to attract a diverse range of animal pollinators, predominantly insects, to facilitate their reproductive cycle. The metabolism of floral scent components, comprising small molecules and volatile chemicals, entails a complex interplay of physiological and biochemical processes. Understanding this intricate mechanism is crucial for unraveling the intricate biology behind floral scents in ornamental plants[29]. With further research on plant secondary metabolism, floral scent components and biosynthesis have been continuously elucidated[30]. Mei stands out among other Prunus species due to its ability to produce strong floral scents. Thus, the study of flower fragrance in Mei has drawn plenty of attention recently. A study conducted in Wakayama Prefecture, Japan, identified 22 components of Mei floral scents through methanol extraction under reflux[31]. However, it was noted that the components extracted by the solvent might not fully reflect the actual aroma components released by Mei. To overcome this limitation, researchers employed headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS) to identify the floral scent components of selected Mei cultivates[32], and the results showed that benzaldehyde and benzyl acetate were the key components affecting the aroma intensity of Mei, with their relative contents are 75% and 90.36%, respectively. The relative content of benzyl acetate in P. sibirica was only 0.06%, indicating that benzyl acetate is a characteristic volatile component of Mei. Except for benzaldehyde and benzyl acetate, many unique components, including eugenol, benzyl alcohol, cinnamyl alcohol, cinnamyl acetate were discovered in various Mei cultivars[11,33]. Generally, the different types and contents of compounds released by Mei are the fundamental reasons for the difference in flower scents of Mei cultivars. In one example, benzyl acetate and eugenol made up the majority of the floral volatiles in cultivars with white flowers[34], like 'Fuban Lve', 'Zaohua Lve', 'Subai Taige' and 'Zao Yudie', whereas only 'Fenpi Gongfen', 'Jiangsha Gongfen', and 'Fenhong Zhusha' (pink flowers) synthesized cinnamyl alcohol and cinnamyl acetate. The endogenous extract of the interspecific hybrids in Mei contained less benzyl alcohol, but more benzyl benzoate, which had a competitive inhibition on the production of benzyl acetate, which may result in the difference in characteristic scent between Mei and its interspecific hybrids[33]. The complex biosynthesis of scents compounds from Mei resulted in a wide variety of volatile chemicals with various levels of concentrations. There were notable variations in the endogenous content and volatilization of main components of 'Sanlun Yudie' during the whole flowering stage. In the bud stage, all volatiles were low, and no eugenol was detected. Benzaldehyde had the highest volatility at the end of flowering, benzyl alcohol and benzyl acetate had the highest volatility at full flowering stage, and eugenol had the highest volatility at fading stage. The content of benzaldehyde was the highest at bud stage, benzyl alcohol and eugenol at fading stage, and benzaldehyde acetate at full flowering stage[32]. In addition, previous studies indicated that mostly emit benzenoid chemicals[35]. Subsequent studies further divided the stamens into anthers and filaments and found that filaments primarily emitted benzyl acetate, while anthers primarily released benzaldehyde[36].

      Based on genome-wide analysis and RNA sequencing, a substantial number of flower scent-related genes have been discovered (Table 1). A comparison of gene expression differences between the two flowering periods (developed bud and squaring flower) of the 'Sanlun Yudie' revealed 6,954 DEGs and 595 transcriptional regulators included (TFs) of 76 TF families. Under the influence of phenylalanine ammonia-lyase (PAL), the essential protein in the synthesis of phenylpropane and the benzene ring, phenylalanine produces trans-cinnamic acid[37]. The P. mume genome contained three PmPALs, and PmPAL2 might contribute to synthetic aroma compounds[38]. P450 proteins were found to be particularly abundant during Mei's blooming stage, and two P450 genes were prominently shown in the DEGs that were upregulated[38]. The short-chain dehydrogenases/reductases (SDR) family was closely related with the formation of benzyl alcohol. A total of 147 SDR genes were identified in P. mume genome, and nine candidate genes were significantly expressed in flowers[38,39], suggesting that they might be associated with the synthesis of benzaldehyde and benzyl alcohol in Mei. The MYB family gathered the most 50 TF, followed by 42 basic helix loop-helix (bHLH), and 35 NAC. A total of 36 TFs specifically expressed in flowers were dispersed over 18 TF families[40], including six MYB-related, six MYB, three NAC, and so on. The MYB family was found during the synthesis of flower scent. At present, four MYB TFs (MYB1/2/3/4) from Mei have been identified and described, and the expression levels of three of them increased with the blooming of flowers[18]. In addition, yeast two-hybrid (Y2HGold) and bimolecular fluorescence complementation (BiFC) assays verified that the metabolism regulation processes involved in floral scents, which affected the expression of downstream genes like 3-deoxy-7-phosphoheptulonate synthase (PmDAHPS), arogenate dehydratases (PmADT), PmPAL, CoA ligase/acyl activating enzyme (PmCNL/AAE). Fourty four PmBEATs genes were found in the P. mume genome[11]. PmBEAT34/36/37 were highly expressed in flowers and their highest expression was observed at the blooming stage. Mei flower cell ability to synthesize benzyl acetate could be influenced by the expression levels of PmBEAT36/37. PmBEAT34/3/37 all had benzyl alcohol acetyltransferase activity in vitro[11]. Coniferyl alcohol acetyltransferase (CFAT) is a crucial substrate for the synthesis of eugenol, which catalyzes the conversion of coniferyl alcohol into coniferyl acetate. The 90 PmBAHD (including 44 PmBEAT family) genes were screened from the whole genome and phylogenetically divided into five major groups. PmBAHD67-69 might have a role in the metabolism of floral scents[41]. Two CFAT genes (PmCFAT1 and PmCFAT2) were cloned, and bioinformatics analysis and expression profiling suggest that PmCFAT1 may be crucial for eugenol biosynthesis but not PmCFAT2[34]. DNA methylation is a frequent epigenetic modification and differentially methylated genes (DMGs) were shown to have essential functions in controlling the floral fragrance production of Mei, for instance PmCFAT1a/1c, PmBEAT36/37, PmPAAS3, PmBAR8/9/10, and PmCNL1/3/5/6/14/17/20[11,32,34,42]. O-methyltransferase (PmOMT) may regulate the formation of methyl eugenol and is highly expressed in flower organs in Mei. Three eugenol synthase genes were cloned by RT-PCR from the blooming flowers of 'Sanlun Yudie', named PmEGSI/PmEGS2/PmEGS3 (eugenol synthase genes), respectively. The most significantly expressed PmEGS2 was introduced into petunia (Petunia hybrida 'W115'), which proved that PmEGS2 gene plays a role in the eugenol pathway and participates in eugenol biosynthesis and metabolism[43]. One hundred and thirty ATP-binding cassette (ABC) genes have been found in Mei, classified into eight subfamilies, including 55 PmABCG genes, which were specifically expressed in the flowers[44]. Volatilization of benzaldehyde and benzyl alcohol was substantially connected with PmABCG2/18/26, but negatively correlated with phenylmethyl acetate, and volatilization of benzyl acetate was highly correlated with PmABCG9/13/23. Besides, the study found that PmIAA2/12/15/16 was also involved in the synthesis of benzyl acetate and cinnamyl acetate[45]. These genes, exhibiting high expression levels in various floral organ parts, are believed to have a significant impact on the transmembrane transport of floral components[44]. Through transcriptome analysis and enzyme activity assays, it was demonstrated that PmCAD1 (cinnamyl alcohol dehydrogenase) was identified as having a crucial part in the biosynthesis of cinnamyl alcohol in vitro. These findings shed light on the specific enzymatic pathway responsible for the production of this aromatic compound in Mei[46].

      Table 1.  Functional validation information of flower scent.

      Flower scentGene IDFunction descriptionValidation methodsReference
      PmPAL2Pm030127Involved in phenylpropanoids/
      benzenoids biosynthesis
      HS-SPME-GC-MS Methods[32]
      PmBEAT36/37Pm011009/Pm011010Performs a key part in the biosynthesis
      of benzyl acetate
      Bioinformatics analysis, expression pattern analysis, plasmid construction, subcellular localization, enzyme activity analysis, GC-MS analysis[11]
      PmBARPm012335/Pm013777/
      Pm013782
      Performs a key part in the biosynthesis
      of benzyl acetate, as the key genes responsible for BAR activity
      Integrative metabolite, enzyme activity, and transcriptome analysis, plasmid construction, qPCR validation[42]
      PmBAHD16/25Pm010996/Pm011009Plays an important role in promoting
      the production of benzyl acetate
      GC-MS analysis, bioinformatics analysis, expression pattern and WGCNA analysis, validation of transgenic Arabidopsis plants[41]
      PmIAA2/12/15/16Pm003529/Pm013416/
      Pm013597/Pm020225
      Involved in the synthesis of benzyl
      acetate and cinnamyl acetate
      Bioinformatics analysis, expression pattern analysis[45]
      PmMYBPm015692/Pm021211/
      Pm025253
      Engaged in floral fragrance metabolic control via influencing the expression
      of downstream genes
      GC-MS analysis, bioinformatics analysis, expression pattern analysis, subcellular localization, vector construction[18]
      PmEGSPm012360Involved in eugenol biosynthesisBioinformatics analysis, expression pattern analysis, subcellular localization[43]
      PmCFAT1Pm030674Involved in floral scent metabolismGC-MS analysis, bioinformatics analysis, expression pattern analysis, subcellular localization[34]
      PmABCG2/18/26Pm001070/Pm022014/
      Pm029602
      Positively linked with benzaldehyde and benzyl alcohol volatilization ratesBioinformatics analysis, expression pattern analysis, GC-MS analysis of volatile components[44]
      PmABCG9/13/23Pm011453/Pm012323/
      Pm026080
      Positively linked with benzaldehyde and benzyl alcohol volatilization ratesBioinformatics analysis, expression pattern analysis, GC-MS analysis of volatile components[44]
      PmCAD1/2Pm021215/Pm021214Play roles in cinnamyl alcohol synthesisGC-MS analysis, bioinformatics analysis, expression pattern analysis, real-time fluorescence quantitative PCR, vector construction[46]
    • The color of a flower is a crucial factor that contributes to its ornamental value. In the case of the Prunus species, including Mei, the flowers exhibit a range of colors, primarily purplish-red, pink, pure white, greenish-white, yellowish, and compound colors[47]. The flower color phenotypes of the petals of several varieties of Mei at different developmental stages were determined using colorimetric and chromameter measurements, it was found that the brightness and chromaticity of the flower color of different varieties of Prunus were mainly affected by the value of a*. As the flower color deepened from white to purplish-red, the value of a* (hue) gradually increased. According to the significance of the relationship between the flower color parameter brightness L* and the chromaticity c* and the chromaticity a*, this was related to the anthocyanin content of Mei blossoms, and anthocyanidin glycosides are the key components of flowers that exhibit colors such as pink, red, purple and blue. The main pigments of Mei safflower were determined as anthocyanins and flavonoids by high performance liquid chromatography (HPLC)[48]. The main components of anthocyanin glycosides in the red line of Mei were Cy3GRh, Pn3GRh, and cornflower-3-O-glucoside (Cy3G), and only the white line did not contain any anthocyanin glycosides, which were colorless or flavonoids determining the white color[18].

      Structural genes and transcription factors like MYB, bHLH, and WD40, which may compose the MYB-bHLH-WD40 (MBW) protein complex and participate in the biosynthesis of secondary compounds such as anthocyanins, are typically responsible for controlling the expression of anthocyanin synthesis genes[49]. Among them, MYB is a crucial gene regulating floral color. It affects the expression of structural genes PAL, CHS (chalcone synthetase), F3'5'H (flavonoid-3',5'-hydroxylase), and ANR relevant to flower color and encourages the accumulation of anthocyanins and flavonoids[50]. Major anthocyanin synthesis-related genes were isolated and characterized in the P. mume genome (Supplemental Table S1), and those validated included transcription factors such as PmMYB and PmWD40-48[22,51,52], which are involved in anthocyanin synthesis, and structural genes such as PmF3'H and PmUFGT3 (flavonoid glycosyltransferase), which contribute to red pigment formation[18,48,53]. In addition, the structural genes PmDFR (dihydroflavonol reductase) and PmANS (anthocyanin synthase) may be target genes for the transcription factor PmMYBa1[22]. Similarly, flavonoid and anthocyanin content were found to be the main cause of stem color differences in the study of xylem color traits[54]. In addition, Pm009966, Pm011003 (PmBAHD), Pm011258, Pm017164, Pm019289, Pm020893, Pm025210 (PmCYP450), Pm000414, Pm001802, Pm004453, were identified in the differently methylated regions of red and white petals of Mei, Pm020721, Pm027780, Pm013365 (PmABC), and 13 DEGs as key candidate genes[39].

    • The morphology of flowers serves as one of the fundamental criteria for classifying different varieties of Mei. The development and morphology of floral organs are intricate processes influenced by various factors and their interactions. The diverse range of floral organ morphology and number in Mei has resulted in the emergence of distinct varieties characterized by features such as monopetalous (single-petaled), double-petalous (double-petaled), prolification (abnormal proliferation), flying petalous (petals with an upward orientation), multiple sepals, multiple pistils, and more[2]. To gain a thorough grasp of the molecular regulatory systems underlying flower morphology in Mei, researchers have conducted studies involving microRNA (miRNA) identification, target gene analysis, expression profiling, and functional characterization (Supplemental Table S2). These investigations have expanded our knowledge beyond the post-transcriptional level, shedding light on the intricate processes governing Mei flower development[55]. Through Gene Ontology (GO) analysis, researchers have identified several key microRNAs and their potential target genes involved in regulating various processes related to Mei flower development. Transcription factors, such as those belonging to the GRAS/HAM and Auxin response factors (ARF) families, have been identified as important targets of microRNAs in Mei flower development and played crucial roles in regulating gene expression and coordinating various aspects of flower development, including organ formation, patterning, and differentiation. Additionally, metabolism-associated genes, such as β-glucosidase, acyltransferase, α-1,4 glucan phosphorylase L isoform, and pyruvate dehydrogenase E1, have been identified as targets of miRNAs in Mei flower development. These participate in various metabolic pathways, including carbohydrate metabolism, lipid metabolism, and energy production, which are essential for supporting flower growth and development. The GO analysis of miRNA-regulated genes in Mei flower development provides valuable insights into the molecular mechanisms underlying these processes. By understanding the roles of specific microRNAs and their target genes, researchers can further elucidate the complex regulatory networks that govern flower development in Mei and potentially manipulate these pathways to improve desired traits in cultivated varieties[56,57]. Among them, the transcription factor GARS identified a total of 46 genes in Mei validation of the subfamily DELLA (regulating GA signaling) revealed that PmDELLA is involved in the gibberellin signaling pathway controlling the breaking of dormancy and germination of seeds in Mei[55]. Exogenous gibberellin treated 'Mingxiao Fenghou' branches released from dormancy after 20 d with more than 50% sprouting rate, and the treated branches released from dormancy 31 d earlier than the natural dormancy. Based on the ABCDE model, MADS-box genes involved in flower structure formation were identified and categorized in the P. mume genome. The genes that fulfill the function of class A genes are subfamily Apetala1/Fruitfull (AP1/FUL) of three members (PmAP1, PmFUL1, and PmFUL2)[58]. Class B genes contain the subfamily Apetala1/Pistillata (AP3/PI), with PmAP3 playing a role in gynoecium development, and PmPI and PmAP3-2 in petal and stamen development. Class C genes (PmMADS15/PmAG) are associated with stamens and gynoecium, and class D genes (PmMADS03) are associated with gynoecium[5860]. PmSEP2 (sepallata) and PmSEP3 are associated with petals, stamens, and pistils, and PmSEP1 and PmSEP4 control sepals, suggesting a function for their E-class genes. Besides, two SOC1 (suppressor of overexpression)-like genes (PmSOC1-1 and PmSOC1-2), and one LFY (LEAFY)-like gene (PmLFY1), as well as the SVP (short vegetative phase) gene (PmSVP1), were also involved in flower organ morphology[6163]. It has been found that the miR156-PmSBP1-PmSOC1s pathway was discovered to be involved in the controlled blooming[64]. In addition, Y2HGold confirmed that there are protein-protein interactions between different classifications in the MADS-box genes of the P. mume genome[18,65]. Twenty candidate genes, including the hub genes PmAP1-1 and PmAG-2, for the Mei double flower trait were screened out in a study comparing the morphological differences between the floral organs of single and double flower cultivars. Interestingly, Mei's double flower feature frequently coexisted with petaloidal stamens, multiple carpels, and an increase in the overall number of floral organs[66]. The recently created molecular markers can be utilized to identify double bloom of Mei early on and set the stage for future advancements in the breeding effectiveness of double flower of Mei[67].

    • The weeping trait is a distinctive tree-like structure found in woody plants, where lateral branches naturally droop and grow downward. In weeping varieties of Mei, the branches and trunks exhibit a drooping form, adorned with naturally decorated, colorful flowers. With their elegant tree shape, these varieties have significant value and significance in both floral display and as ornamental trees for tourism and horticultural applications. The candidate region for weeping in Mei was identified as 1.14 Mb by QTL, containing 159 predicted genes. The development of SLAF-seq (Specific-locus amplified fragment sequencing) markers was conducted using the F1 population of the hybrid offspring between 'Six Petals' and 'Pink Terrace weeping'. Through QTL fine mapping, the candidate genes for the weeping trait of Mei were localized to the 10.54 to 11.68 Mb region on chr7 of Mei. Furthermore, 18 candidate genes likely related to the control of the weeping trait (Supplemental Table S3) were further screened[26]. Lignin biosynthesis can affect fiber development and thus lead to differences in stress wood structure, and the lignin content of the proximal surface of weeping Mei branches was higher than that of the distal surface, in contrast to that of straight Mei[68]. Nine genes involved in cell wall formation and development and lignin synthesis, selected from them, including cellulose synthase CSL family members Pm024150 and Pm024152, dextranase Pm024254, which contributes an essential part in cell wall alteration, Pm024195, and Pm024255, which are participate in cell wall formation and assembly, as well as those present in secondary cell wall biosynthesis in the growth factor metabolic pathway is positively regulated by the factor growth hormone-inducible protein 5NG4-like (Pm024277)[69]. Related genes whose lignin biosynthesis can regulate tissue secondary cell wall lignification include, Pm024136, which may be involved in xylan metabolism, cinnamyl alcohol dehydrogenase 1 (Pm024278), Pm024136, and the NAC family gene NAC conserved domain protein 43 (Pm024260)[70]. Among them, Pm024260 has been shown to be linked to the development of secondary cell walls in Mei branches[71]. Furthermore, it was found that the TAC1 (Tiller Angle Control 1) gene was involved in the regulation of branching or tillering angle of plants, PmTAC1 (Pm018391) possessed the typical structural domains of the IGT family, with the highest expression in the stems and the content was much higher in the annual weeping branches than that in the straight branching varieties, and there were differences in the proximal and distal axes of the two kinds of branches. The sequence of the coding region of PmTAC1 showed no differences between the two kinds of branches, but the promoter had sequence differences[72].

      Annotation of candidate regions for the weeping trait identified nine genes which affect gene expression at the transcriptional or post-transcriptional levels. Among them, the 26s proteasome (Pm024160) plays a part in the balance between cell elongation and cell differentiation during branch development. The transcription factor 22 protein NIN like Protein7 (NLP7) plays a part in the regulation of nitrate assimilation and signaling processes. The transcription factor bHLH155 (Pm024214) is involved in root development[73]. The transcriptional activator of the growth regulator 8 (Pm024257), which controls cell elongation in meristematic tissues. RPB1 (Pm024270/Pm024271/Pm024275/Pm024123), a DNA-directed RNA polymerase II subunit, all of which are regulatory genes that can cause differential expression of downstream gene regulatory networks[28]. In addition, branch development and plant architecture are regulated by the plant HD-Zip III transcription factor, which is a key transcription factor controlling the meristematic tissue's formation and maintenance. The results of transgenic Arabidopsis thaliana GUS tissue staining revealed that PmHB5 gene was primarily expressed in meristematic tissues, vascular tissues, and other parts of the plant where cell growth and differentiation were active, and it was hypothesized that it had an impact in the regulation of cell differentiation and branch lignification in stems of Mei[74].

      The external factors that altered the angle of inclination of Mei branches were in turn, indole-3-acetic acid (IAA), phototropic growth, and gibberellic acid (GA3). The weeping phenotypic traits of Mei were mostly linked to the phenylpropanoid biosynthesis pathway, the phytohormone signaling pathway, and the pathway of starch and sucrose metabolism. In the phenylpropanoid biosynthesis pathway, PER-like genes involved in the process of peroxidase action are associated with vertical traits and exogenous GA3, and such genes are also key genes affecting the weeping traits[75]. Vertical traits also have candidate genes associated with key enzyme activities. Marker437413 (Pm024200), which is involved in auxin transport and metabolism, has been shown to promote cell elongation by ethylene, and thus may be involved in cell elongation due to the candidate genes Pm024219, a serine/threonine protein kinase, with no lysine kinase (WNK8), and Pm024247, encoding a chytridiomycin-like protease[76].

      RNA sequencing and comparison of DEGs in straight and weeping Mei's bud and stem tissues were used to identify genes associated with IAA (Pm013243, Pm005112, Pm007046, Pm020838, Pm024306, Pm03020, Pm012502, Pm021243, Pm005182), GA (Pm004966, Pm010085, Pm011672, Pm01992, Pm011163) and some key genes related to lignin (Pm012986, Pm027089, Pm027248)[77]. Combined with QTL analysis, the 9.69-10.65 Mb region on chromosome 7 where the five QTLs associated with weeping traits were located was deemed a highly dependable region linked to weeping traits. Two significant markers markker313919 (Pa7:11324965) and markker437413 (Pa7:11037771) associated with the core SNP (Pa7_11182911) for weeping trait were found based on GWAS and QTL[78]. The main QTL was localized in the 11.03−11.32 Mb interval, also located on chr7. In addition, it was found that the two regions localized to chr7 strongly interacted with each other[78]. Finally, the gene closest to the core SNP was identified as Pm024213, which was found to be highly up-regulated and specifically expressed in weeping by GWAS analysis from 39 candidate genes[18]. In addition, Pm024213 contains a Trx structural domain that may regulate weeping traits via growth hormone-mediated gravity-sensing or light-responsive processes[78].

    • One of the key things limiting Mei cultivars from being bred and promoted at the moment is abiotic stress. The majority of the abiotic stresses that Mei encountered were related to freezing, although research has also been done on how Mei responded to stress from salt, drought, and hot temperatures. As a result, understanding the mechanism underlying abiotic stress is essential to boosting breeding efficiency going forward.

    • Mei originated in southwest China and the Yangtze River basin, is a subtropical tree species. However, in the northern region of China, low temperature has seriously limited the growth of Mei, and few varieties can be applied. The selection and breeding of cold-resistant cultivars of Mei has been an important direction of breeding. Over the years, the cultivation of cold-resistant cultivars mainly focused on the conventional breeding methods such as introduction and domestication, distant cross breeding[5]. Since the 1950s, Chen et al.[2] cultivated a group of cold-resistant of Mei cultivars, such as 'Fenghou', 'Danfenghou' and 'Meimei', by using the introduction and domestication experiments in northern China, and successively carried out regional experiments of different cultivars in Inner Mongolia, Liaoning, Shanxi, Qinghai and Gansu[2,79]. Zhang[5] bred cold-resistant cultivars of Mei such as 'Yanxing' and 'Huahudie', through distant crossbreeding with strong cold resistance Mei relatives, which can withstand low temperatures from −35°C to −25°C. Subsequently, a series of cold-resistant cultivars such as 'Yutaizhaoshui', 'Songchun', 'Zhongshanxing' and 'Shantaobai' were selected and bred through several decades of selection, domestication, cross breeding and distant crossing etc. Most of these cultivars are Apricot Mei Group, which laid the material foundation for the concept of 'Transferring Mei from South to North'. Chen et al.[79] used Apricot Mei Group and Mei cultivars to breed a cold resistant cultivar 'Xiangruibai' with the characteristic scent of Mei, which achieved an enormous advance in cold-resistant fragrant flower breeding of Mei. It is worth mentioning that the broad field cultivation region occupied by Mei has spread 2,000 km from the Yangtze River Basin through many years of multi-point comparative experiments.

      The comparison of physiological changes of Mei can analyze the difference of cold resistance of different cultivars. The cold resistance of 38 Mei cultivars was analyzed, and it turned out this the cold hardiness of apricot Mei series is higher than that of true Mei series[80]. Similarly, the physiological indexes of Mei growing in different dimensions and seasons were analyzed, and it was found that the 'Yanapricot' cultivar had the strongest cold resistance[39].

      With the conclusion of Mei's entire gene sequencing, it is now possible to investigate resistance genes and establish the groundwork for Mei molecular breeding. A vital phase in the upper life cycle of plants, dormancy is an adaptive reaction that allows plants to withstand harsh environments, like cold temperatures[81]. The reaction to cold stress and dormancy was modeled molecularly[82] using the interaction between PmDAM and PmCBF. It was discovered that low temperatures triggered the production of PmCBF, and the build-up of CBF encouraged the development of PmDAM, causing flower buds to go into dormancy[82]. Candidate TFs and target genes that can control Mei dormancy by adjusting endogenous hormone content in response to environmental cues were effectively screened in the coexpression network of genes linked to flower bud dormancy[83]. Among them, ABRE binding factor PmABF2, PmABF4, and PmSVP changed Abscisic acid (ABA) content to govern bud dormancy[83]. Furthermore, it was shown that PmSOC1-2, which interacts with PmDAM, regulates floral bud dormancy via ABA regulation[84]. At present, the research on the molecular mechanism of Mei blossom cold resistance mainly focuses on CBF (C-repeat binding factors), ICE1 (Inducer of CBF expression1), ERF (ethylene responsive transcription factor), LEA, DAM, WRKY and other gene families[7,82,8587]. Cold stress, which includes chilling (cold temperatures of above 0 °C) and freezing (below 0 °C) stress, is a significant factor limiting plant growth, development, and geographical distribution[88]. Cold acclimation is a protective strategy promoting plant tolerance and resistance to cold stress that is controlled through both CBF-dependent and CBF-independent pathways[89]. Six PmCBFs genes were cloned from Mei, and all of them have been shown to be triggered by cold stress, and the function of transgenic plants was verified[90]. CBF and DAM are the key genes in Mei that respond to cold and dormancy respectively, and the possible interrelationships surrounding the CBF and DAM genes, as cold acclimation and dormancy are closely linked. In particular, the interaction among PmCBFs with PmDAM reveals the molecular mechanism behind cold-response pathway and dormancy regulation in Mei growth[82]. ICE1, a member of the bHLH transcription factor family, might activate AtCBF3 and AtCOR genes in reaction to low temperature[85,91]. Overexpression of PmICE1 increased cold resistance in Arabidopsis compared with control[92]. Meanwhile, other bHLH transcription factors may influence cold tolerance of Mei. There were 95 PmbHLH genes found in the P. mume entire genome, which were grouped into 23 subfamilies. Through investigation of transcriptome and qRT-PCR data, PmbHLH4/6/25/28/38/40/57 was discovered playing a major part in resisting low-temperature stress[93,94]. The overexpression of PmBBX32 gene reduced the damage to Arabidopsis and may improve transgenic plant cold resistance by increasing antioxidant enzyme activity and proline content[95]. Validation of transgenic tobacco showed that overexpression of PmPUB1, PmPUB3 (Plant U-Box E3 ligases) and PmWRKY18 genes increased cold resistance[87,96]. A total of 30 late embryogenesis abundant (LEA) genes were identified on a genome-wide level using the Hidden Markov Model (HMM), four (PmLEA10/19/20/29) of these genes were involved in plant responses to cold[86]. In addition, PmWRKY18 and PmLEA8/19/20 were induced to be expressed by Atco exogenous ABA and may be involved in ABA-dependent cold signaling regulatory pathways[86,87]. Freezing tolerance genes have been discovered in Mei by RNA-seq and ATAC-seq (assay for transposase-accessible chromatin using sequencing) analysis. Cold-shock protein CS120-like (PmCSL) expression also considerably significantly up-regulated, meanwhile the chromatin opening of PmCSL was markedly increased[97]. The freezing resistance of transgenic Arabidopsis plants was markedly enhanced by overexpressing PmCSL. Afterward, a large number of genes associated with cold resistance were found in P. mume genome, like 13 HDACs (histone deacetylases)[98], 49 bZIP (basic leucine zipper) transcription factors[99], 113 NAC transcription factor genes[100], 17 SWEET (sugars will eventually be exported transporter)[101], 58 WRKYs[87,102], 16 CIPKs (serine/threonine protein kinase)[103], 11 MAP kinases (mitogen-activated protein kinase, MPKs) and seven MAPK kinases (MKKs)[104], and Table 2 contains additional functional genes and information[105109]. Gene structures, phylogenetic relationships, cis-acting elements, and expression patterns in reactions to cold treatment were all intensively investigated in order to obtain insights into the mechanisms underlying cold response in woody plants. These investigations have significantly enhanced our comprehension of the roles of gene families involved in cold tolerance. By elucidating the genetic basis of cold response, these studies have provided valuable information for the development of molecular breeding programs in woody plants.

      Table 2.  Functional validation information of cold resistance.

      Cold resistanceGene IDFunction descriptionValidation MethodsReference
      PmCBF1/2/3/4/5/6Pm023769/Pm023772/
      Pm023773/Pm023775/
      Pm023777/Pm027913/
      Key TF that responses to cold signalqRT-PCR, gene cloning and Yeast 2 Hybrid assays, BiFC assays, promoter cloning and Yeast 1 Hybrid assays[82]
      PmICE1PmCBF express levels are increased in response to a low temperature signalBioinformatics analysis, expression pattern analysis, subcellular localization, vector construction, Arabidopsis transformation, and low-temperature stress experiments[92]
      PmLEA10/29Pm026684/Pm006114Besponse to cold stressGene expression analysis, Tobacco transformation and stress tolerance analysis, Relative Water Content (RWC), protein assay and analysis, statistical approach for MDA and REL[86]
      PmLEA19/20Pm020945/Pm021811Response to cold stress; participate in ABA-dependent pathwayGene expression analysis, Tobacco transformation and stress tolerance analysis, Relative Water Content (RWC), protein assay and analysis, statistical approach for MDA and REL[86]
      PmRSPm027594/Pm025896Diminish the negative effects of coldGene cloning, expression pattern analysis, subcellular localization, transformation of Arabidopsis thaliana, cold resistance analysis, transformation of Mei[107]
      PmBBX32Pm013051Diminish the negative effects of coldExpression pattern analysis, transformed Arabidopsis thaliana, low-temperature stress treatment, physiological index determination[95]
      PmCIPK5/6/13Pm001690/Pm018300/
      Pm008498
      Modulating the stress response to coldBioinformatics analysis, expression pattern analysis, qRT-PCR[103]
      PmNAC11/20/23/40/
      42/48/57/59/60/61/
      66/82/85/86/107
      Pm001403/Pm005783/ Pm006470/Pm011234/
      Pm011603/Pm012630/
      Pm015876/Pm017550/
      Pm018292/Pm018442/
      Pm019659/Pm024558/
      Pm025184/Pm025307/
      Pm025184/Pm025307/
      Pm028721
      Involved in the cold-stress responseBioinformatics analysis, expression pattern analysis, qRT-PCR[100]
      PmbHLH4/6/25/28/ 38/40/57Pm002111/Pm002283/
      Pm008898/Pm016406/ Pm018355/Pm023237
      Play a critical part in the resistance to low temperature stressBioinformatics analysis, expression pattern analysis, qRT-PCR[93,94]
      PmPUB1/3Pm006753/Pm009248Play a significant part in the regulatory network connected to low temperature stressBioinformatics analysis, expression pattern analysis, qRT-PCR, overexpression of tobacco, low-temperature treatment, physiological index measurement[87]
      PmWRKY18Pm005698Play a significant part in the regulatory network connected to low temperature stress, sensitive to ABA treatmentBioinformatics analysis, expression pattern analysis, qRT-PCR, overexpression of tobacco, low-temperature treatment, physiological index measurement[87]
      PmWRKY57LOC103321497Function in improving cold tolerance of plantsCloning and sequence analysis, subcellular localization, transformation of A. thaliana, determination of plant physiological index, expression analysis of genes[102]
      PmSOD3Pm003436Had important regulatory roles in cold acclimation processPhysiological index determination, section observation, tissue browning, ion leakage rate, infrared thermal imaging technology, and freeze thaw detection sensors[109]
      PmPOD2/19Pm000967/Pm022119Had important regulatory roles in cold acclimation processPhysiological index determination, section observation, tissue browning, ion leakage rate, infrared thermal imaging technology, and freeze thaw detection sensors[109]
      PmNCED3/8/9Pm005153/Pm011164/
      Pm016267
      Significant role in the plant's response to cold stressBioinformatics analysis, expression pattern analysis, qRT-PCR[108]
      PmCSLSignificantly improved the freezing tolerance of transgenic plantsBioinformatics analysis, expression pattern analysis, ATAC sequencing, gene cloning and gene expression analysis, plant transformation and low temperature treatment[97]
      PmHDAC1/6/14Pm020717/Pm024325/
      Pm012683
      Significantly respond to cold stressBioinformatics analysis, expression pattern analysis, qRT-PCR[98]
      PmbZIP 12/31/36/41/48Pm005288/Pm020080/
      Pm021804/Pm025001/
      Pm029028
      Responses to low-temperature stressBioinformatics analysis, expression pattern analysis, qRT-PCR[99]
      PmSWEET1/12/13/14Pm007697/Pm022696/
      Pm024167/Pm024554
      Responses to cold stressBioinformatics analysis, expression pattern analysis, qRT-PCR[101]
      PmCDPK14Pm026757Play an essential role in resisting low temperature stressBioinformatics analysis, expression pattern analysis, qRT-PCR, compare between two genomes of Mei[105]
      PmMAPK3/5/6/20Pm000966/Pm023935/
      Pm027774/Pm014593
      Significantly respond to cold stressBioinformatics analysis, expression pattern analysis, qRT-PCR, compare between two genome databases of Mei[104]
      PmMAPKK2/3/6Pm027015/Pm015648/
      Pm027289
      Significantly respond to cold stressBioinformatics analysis, expression pattern analysis, qRT-PCR, compare between two genome databases of Mei[104]
      PmRCI2sPm027750/Pm003262/
      Pm003263
      Significantly induced by low temperatureBioinformatics analysis, expression pattern analysis, qRT-PCR[106]
    • At present, a great deal of research has revealed that abiotic stress affects woody plants at many phases of their growth and development, such as seed germination, growth, flowering, and fruiting[110]. Drought stress was one of the main abiotic factors that Mei had to deal with; as a result, the plants became shorter and had fewer leaves[111,112]. Salt stress mostly reduced germination rate and altered bloom yield and branch length[113]. By subjecting annual branches of Mei to drought treatment, drought-responsive functional proteins PmCCD1/4/8 (carotenoidcleavage dioxygenase)[114] and PmLEA10/29[86], as well as PmTPS2/6 (Trehalose-6-phosphate synthase)[115] involved in trehalose synthesis, were discovered. By treating cultivars with varying degrees of drought tolerance, the important genes N-acetyl-serotonin methyltransferase (PmASMT1) and tryptophan 5- hydroxylase (PmT5H1), which were highly responsive to the melatonin production pathway were also found, and the corresponding physiological indices were ascertained by creating overexpressed plants[112]. Furthermore, a multitude of transcription factors that respond to drought were found, including PmbZIP5/29/35, PmbHLH35, PmWRKY2-1/2-2, PmZAT12 (zinc finger of arabidopsis thaliana12), and PmTALE1/3/6/13 (three amino acid loop extension)[116120]. The genes PmbZIP, PmbHLH, PmWRKY, PmLEA, and PmCIPK that were previously found to be involved in freezing stress are also implicated in drought stress, indicating that several of the functional genes mentioned above respond to more than one abiotic stress. PmCCD1/4/8, PmZAT12, PmTALE1/3/6/13, and PmbZIP5/29/35 was also responsive to salt stress found in drought stress[99,114,116,119,120]. It is possible to identify and confirm the essential genes PmCIPK21, PmbHLH35, PmMYB, and PmNF-YA2/YB3 during salt stress by monitoring changes in physiological markers, such as Superoxide dismutase (SOD) and malondialdehyde (MDA)[117,121]. Apart from investigating the mechanism of salt stress on Mei using different bioinformation techniques, it was discovered that grafting could enhance salt resistance in practical breeding[122]. Grafted seedlings have the ability to greatly boost leaf photosynthesis in comparison to self-rooted seedlings. Furthermore, the majority of studies on temperature extremes have concentrated on low-temperature stresses. Nevertheless, when it comes to high temperature stress, it is discovered that high temperature has an impact on both the duration of the watching period and the overall viewing effect. Presently, PmHSP17.9 of the heat hormone protein (HSP) has been successfully screened and cloned[123], and its role in high heat has been successfully established through changes in physiological indexes following abiotic stress treatment.

      Although the aforementioned genes for heat, drought, salt, and cold tolerance have been identified (Supplemental Table S4), and the corresponding genes have been obtained through preliminary cloning, the molecular mechanisms underlying resistance to abiotic stress remain unclear, and there is currently no ideal genetic transformation system. Furthermore, further research is required to understand how different stress factors interact with one another.

    • Mei, as a woody plant with a lengthy cultivation history, holds not only high ornamental value but also a unique cultural significance among many ornamental plants due to its special flowering season. The trees are naturally distributed and cultivated mainly in the Yangtze River basin and the regions south of the Yangtze River in China. 'Trekking in the snow in search of Mei' is a distinctive garden landscape in the Jiangnan region[79]. After decades of continuous research and practice, the number of Mei varieties has exceeded 450, classified into 11 species groups[4]. Several varieties of Mei can be grown in the open air in the Yellow River basin and areas north of it in China. Although some progress has been made in Mei breeding, the existing varieties can no longer meet the demands of today's horticultural market.

      Currently, in terms of ornamental traits, Mei varieties with excellent cold resistance often lack noticeable floral fragrance. Weeping varieties and the Tortuous Dragon group are commonly planted in field settings. The long-term goal of Mei breeding is to cultivate new varieties with diverse floral styles, distinctive floral fragrances, colorful flowers, and strong cold resistance[124]. Thus, improving the ornamental quality while breeding Mei germplasm that is drought-resistant, cold-resistant, salt-resistant, heavy metal-resistant, moisture-resistant, and waterlogging-resistant will be another important research direction in ornamental plant breeding. Hybrid breeding, selective breeding, and bud mutation breeding are the main methods employed in breeding Mei. However, the limitations of traditional methods are gradually becoming apparent due to technological advancements and the high heterosis of Mei. Therefore, adopting new technologies to breed new high-quality varieties is also a long-term goal that needs to be pursued (Fig. 2).

      Figure 2. 

      Timeline of research on P. mume genomics and prospects.

    • Mei resources have irreplaceable ornamental and humanistic values in the process of human social development. Tissue culture research of woody plants enables asexual reproduction of forest trees and preservation of excellent germplasm resources, providing a foundation for establishing a genetic transformation system. Mei currently needs to establish a set of perfect expression system, and utilize the efficient genetic transformation system to further clarify the function of specific genes. Several previous studies have documented transient transformation of Mei protoplasts and genetic transformation systems for immature or mature cotyledons[125,126]. These transformation systems serve as references for optimizing experimental settings and improving the efficiency of genetic transformation. However, compared to functional validation of other woody plants, Mei is currently limited to model plants or even herbaceous model plants. In recent years, the gene editing technologies led by CRISPR/Cas have been developing rapidly, and the successful establishment of plant genetic transformation systems is a necessary prerequisite for the application of such technology, highlighting the evident importance of establishing a transformation system.

    • Given the rapid advancement of genomics and its application in the expansion of new Mei varieties, analyses at multiple levels such as signal transduction, transcriptomics, proteomics, metabolomics, etc., have gradually revealed the mechanisms underlying the important ornamental characteristics of Mei. However, these studies have been limited to model plants and some crop species, with relatively few studies conducted on woody plants. With the third generation of improved single-molecule sequencing technology (e.g., HiFi reads (high-fidelity reads)) and high-C aided assembly technologies, coupled with the decreasing cost of gene sequencing, genomic studies on various species of Mei have become feasible. In addition, there is still a long-term challenge in terms of the lack of sufficient mutational resources for molecular cloning, functional characterization, and annotation of genes responsive to adversity stress in Mei[127]. In recent years, the combined application of IGS and CRISPR/Cas9 technologies has provided broad prospects for future functional analysis in Mei[128]. This technology has been researched and applied in this plant regarding stress tolerance, regulation of flowering time, and alteration of tree shape. However, since Mei has not yet established a complete system of regeneration by histoculture and a stable system of genetic transformation, it is not possible to utilize biotechnology such as gene editing to carry out genetic improvement. Therefore, it is necessary to develop and improve the genetic transformation system of Mei in order to expand the scope of application of the CRISPR/Cas system. In the future, we will enhance the quantity and quality of the genome, jointly resequencing the transcriptome, proteome, and metabolome for multi-omics analysis, and construct a gene regulatory network to fully understand the regulation and interaction between different genes. This will help us comprehend the molecular mechanisms underlying the growth, development, and formation of important traits in Mei. Subsequently, relying on the mature genetic transformation system and the application of new technologies such as CRISPR/Cas9, we will continue to breed new varieties with ornamental traits and excellent resistance of Mei. Continued advancements in these techniques are expected to further propel Mei research and contribute to the sustainable utilization of Mei resources.

    • The authors confirm contribution to the paper as follows: visualization: Fan D, Wen Z, Liu X; methodology: Miao R; writing – review & editing and formal analysis: Sun L; writing – original draft: Fan D, Miao R, Lv W, Meng J, Cheng T, Zhang Q. All authors reviewed the results and approved the final version of the manuscript.

    • Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

      • This study was supported by the Fundamental Research Funds for the Central Universities (QNTD202306); Forestry and Grassland Science and Technology Innovation Youth Top Talent Project of China (No. 2020132608) and Beijing High-Precision Discipline Project, Discipline of Ecological Environment of Urban and Rural Human Settlements.

      • The authors declare that they have no conflict of interest. Lidan Sun is the Editorial Board member of Ornamental Plant Research who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and the research groups.

      • # Authors contributed equally: Dongqing Fan, Runtian Miao

      • 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/.
    Figure (2)  Table (2) References (128)
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    Fan D, Miao R, Lv W, Wen Z, Meng J, et al. 2024. Prunus mume genome research: current status and prospects. Ornamental Plant Research 4: e006 doi: 10.48130/opr-0024-0004
    Fan D, Miao R, Lv W, Wen Z, Meng J, et al. 2024. Prunus mume genome research: current status and prospects. Ornamental Plant Research 4: e006 doi: 10.48130/opr-0024-0004

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