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Genome-wide identification and characterization of the Lateral Organ Boundaries Domain (LBD) gene family in nine Rosaceae species and expression pattern in Prunus mume

  • # Authors contributed equally: Weichao Liu, Xiaoyu Guo

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  • Transcription factors (TFs) encoded by the lateral organ boundaries domain (LBD) gene family are known to control many plant-specific developmental processes. However, the comparative analysis of the LBD gene family in Rosaceae species and its expression pattern in mei remains unclear. Here, we identified a total of 406 LBDs in nine Rosaceae species, including 39 in black raspberry (Rubus occidentalis), 34 in strawberry (Fragaria vesca), 39 in Chinese rose (Rosa chinensis), 42 in peach (Prunus persica), 41 in apricot (Prunus armeniaca), 41 in mei (Prunus mume var. tortuosa), 60 in pear (Pyrus communis), 41 in hawthorn (Crataegus pinnatifida) and 69 in apple (Malus domestica), respectively. The LBDs of nine Rosaceae species were classified into seven major subclasses. The chromosome localization, collinearity analysis, and gene duplication relationship revealed that segment duplication was the main driving force for the amplification of LBDs in the Rosoideae and Amygdaloideae. Ka/Ks analysis suggested most of the LBD gene pairs might be under purification selection. GO and cis-acting elements analysis showed that LBDs may play important roles in many biological processes and could respond to hormones and stresses. RNA-seq data showed that PmLBD17/19/41 genes contained both low-temperature and MeJA response elements and played a significant variation across different geographic locations and periods. PmLBD30, the ortholog of EgLBD29, exhibited an up-regulation followed by a decrease, which is hypothesized to possibly play a role in the formation of a weeping trait in mei. Our studies offer important data about the development of the LBD family in Rosaceae and the subsequent validation of LBDs' functional genes in P. mume.
  • 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 Tables S1 Primers used in this study.
    Supplemental Tables S2 The reference sequences used for gene-specific primers in Prunus mume.
    Supplemental Tables S3 The physicochemical parameters of all LBD proteins in ten Rosaceae genomes.
    Supplemental Tables S4 The number of LBDs of eleven species in each subclass.
    Supplemental Tables S5 Classification of LBD in each subclass.
    Supplemental Tables S6 Segmental duplication events of nine Rosaceae species.
    Supplemental Tables S7 Tandem duplication events of nine Rosaceae species.
    Supplemental Date S1 Ks values of nine Rosaceae species
    Supplemental Date S2 ka/Ks values of nine Rosaceae species
    Supplemental Fig. S1 Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass Ib subfamily members.
    Supplemental Fig. S2 Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass Ic subfamily members.
    Supplemental Fig. S3 Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass Id subfamily members.
    Supplemental Fig. S4 Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass Ie subfamily members.
    Supplemental Fig. S5 Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass IIa and IIb subfamily members. (A) Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass IIa subfamily members. (B) Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass IIb subfamily members.
    Supplemental Fig. S6 Chromosome distribution of LBDs in the Rosoideae. (A) Chromosome distribution of LBDs in R. occidentalis. (B) Chromosome distribution of LBDs in F. vesca. (C) Chromosome distribution of LBDs in R. chinensis.
    Supplemental Fig. S7 Chromosome distribution of LBDs in the Maloideae. (A) Chromosome distribution of LBDs in C. pinnatifida. (B) Chromosome distribution of LBDs in P. communis. (C) Chromosome distribution of LBDs in M.domestica.
    Supplemental Fig. S8 Collinearity of segmental duplication gene pairs of LBDs in the Maloideae. (A) Collinearity of segmental duplication gene pairs of LBDs in C.pinnatifida. (B) Collinearity of segmental duplication gene pairs of LBDs in P.communis. (C) Collinearity of segmental duplication gene pairs of LBDs in M.domestica. The red lines represent the segment duplication (SD) gene pairs of the LBDs.
    Supplemental Fig. S9 The distribution of the main 20 cis-elements in LBD gene promoters.
    Supplemental Fig. S10 Expression pattern of PmLBDs in different developmental stages of flower buds.
    Supplemental Fig. S11 Expression pattern of PmLBDs in different locations and seasons. BJ, Beijing; CF, Chifeng; GZL, Gongzhuling.
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  • Cite this article

    Liu W, Guo X, Zheng T, Li X, Ahmad S, et al. 2024. Genome-wide identification and characterization of the Lateral Organ Boundaries Domain (LBD) gene family in nine Rosaceae species and expression pattern in Prunus mume. Ornamental Plant Research 4: e007 doi: 10.48130/opr-0024-0005
    Liu W, Guo X, Zheng T, Li X, Ahmad S, et al. 2024. Genome-wide identification and characterization of the Lateral Organ Boundaries Domain (LBD) gene family in nine Rosaceae species and expression pattern in Prunus mume. Ornamental Plant Research 4: e007 doi: 10.48130/opr-0024-0005

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Genome-wide identification and characterization of the Lateral Organ Boundaries Domain (LBD) gene family in nine Rosaceae species and expression pattern in Prunus mume

Ornamental Plant Research  4 Article number: e007  (2024)  |  Cite this article

Abstract: Transcription factors (TFs) encoded by the lateral organ boundaries domain (LBD) gene family are known to control many plant-specific developmental processes. However, the comparative analysis of the LBD gene family in Rosaceae species and its expression pattern in mei remains unclear. Here, we identified a total of 406 LBDs in nine Rosaceae species, including 39 in black raspberry (Rubus occidentalis), 34 in strawberry (Fragaria vesca), 39 in Chinese rose (Rosa chinensis), 42 in peach (Prunus persica), 41 in apricot (Prunus armeniaca), 41 in mei (Prunus mume var. tortuosa), 60 in pear (Pyrus communis), 41 in hawthorn (Crataegus pinnatifida) and 69 in apple (Malus domestica), respectively. The LBDs of nine Rosaceae species were classified into seven major subclasses. The chromosome localization, collinearity analysis, and gene duplication relationship revealed that segment duplication was the main driving force for the amplification of LBDs in the Rosoideae and Amygdaloideae. Ka/Ks analysis suggested most of the LBD gene pairs might be under purification selection. GO and cis-acting elements analysis showed that LBDs may play important roles in many biological processes and could respond to hormones and stresses. RNA-seq data showed that PmLBD17/19/41 genes contained both low-temperature and MeJA response elements and played a significant variation across different geographic locations and periods. PmLBD30, the ortholog of EgLBD29, exhibited an up-regulation followed by a decrease, which is hypothesized to possibly play a role in the formation of a weeping trait in mei. Our studies offer important data about the development of the LBD family in Rosaceae and the subsequent validation of LBDs' functional genes in P. mume.

    • LBD genes are transcription factors (TFs) that are peculiar to green plants and may have evolved from charophyte algae. LBD TFs contain a highly conserved lateral organ boundaries (LOB) domain, which is about 100 amino acids. The LOB domain consists of a conserved CX2CX6CX3C zinc finger-like motif at the N-terminal region, a Gly-Ala-Ser (GAS) block in the middle, and leucine zipper-like coiled-coil motif (LX6LX3LX6L) motif[13]. Based on the examination of the LOB domain and phylogenetics, the LBD proteins were categorized into two primary classes, namely Class I and Class II.[3]. The Class I proteins encode a complete LOB domain, while Class II lacks a LX6LX3LX6L motif[1,4].

      With the release of plant genomic information, the LBD gene family has been investigated gradually in several plants. Fourty two LBD family members were identified in Arabidopsis (Arabidopsis thaliana)[1], 35 in rice (Oryza sativa)[5], 44 in maize (Zea mays)[6], 47 in tomato (Solanum lycopersicum)[7], 40 in grape (Vitis vinifera)[8], 58 in apple (Malus domestica)[9], 57 in poplar (Populus trichocarpa)[10], and 46 in Eucalyptus grandis[11]. LBD family members have only been investigated in plants, indicating their crucial involvement in controlling growth and developmental processes particular to plants. LBDs were formerly believed to play a role in the development of various plant organs, including roots, shoot meristems, leaves, flowers, and embryos[12]. For instance, Arabidopsis AtLBD16 and AtLBD29 can regulate lateral root formation[13], AtLBD6 controls stem meristem[14], leaf adaxial identity, and sepal and petal development[15,16], and AtLBD30 is involved in embryogenesis and floral development[17,18] Moreover, recent research has demonstrated that LBDs also have a function in the process of anthocyanin biosynthesis, nitrogen metabolism, secondary growth, shoot-borne root initiation, plant defenses, hormone response and plant regeneration. For example, AtLBD37/38/39 in Arabidopsis and MdLBD13 in apple can negatively regulate anthocyanin biosynthesis and nitrogen uptake and assimilation[19,20]. PtaLBD1 in poplar (Populus tremula × P. alba) regulates secondary phloem development[21], EgLBD37 and EgLBD29 in E. grandis are involved in secondary xylem differentiation and phloem fiber production[11], respectively. Additionally, Class IIIB members can specifically regulate shoot-borne root initiation in angiosperms[22]. The expression level of MaLBD5, derived from the banana species Musa acuminata, was stimulated by treatment with MeJA and exposure to cold stress. This gene is implicated in the enhancement of cold tolerance mediated by MeJA[23].

      The Rosaceae family consists of over 100 genera and can be divided into four subfamilies: Rosoideae, Prunoideae, Spiraeoideae, and Maloideae. Rosaceae plants, including ornamentals, fruit species, aromatic, and medicinal plants, are economically important plant families. In this study, nine representative plants (black raspberry, strawberry, Chinese rose, peach, apricot, mei, hawthorn, pear, and apple) from three traditional subfamilies (Rosoideae, Prunoideae, Maloideae) of Rosaceae were selected to study. The LBD members were first identified in nine plants. Next, we performed phylogenetic analysis, conserved motifs, sequence alignment, chromosome localization, collinearity analysis, and cis-acting element analysis on these genes. Finally, based on transcriptome data and quantitative real-time (qRT)-PCR analysis, we investigated their expression pattern of PmLBDs in different tissues, cold stress, flower bud dormancy release, and plant architecture. Collectively, these investigations will offer fresh perspectives on the evolutionary correlation of the LBD family in Rosaceae and the expression profile of PmLBDs in P. mume.

    • Genome-wide protein data and annotation data of A. thanliana (TAIR 10), P. trichocarpa (v4.0), and nine other Rosaceae species, including R. occidentali s (GDR,v3.0), F. vesca (v4.0.a1), R. chinensis (v1.0), P. persica (v2.0.a1), P. armeniaca (v1.0), P. mume var. tortuous (v1.0), C. pinnatifida (v1.0), P. communis (v2.0), Malus domestica 'HFTH1' (v1.0) were download from the TAIR database (www.arabidopsis.org, accessed on 12 August 2023)[24], the Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 2 September 2023)[25] and the Genome Database for Rosaceae (www.rosaceae.org, accessed on 15 August 2023)[26], respectively.

    • A HMMER search was used to identify the possible LBDs with the LOB domain (PF03195) from the Pfam database 36.0 (http://pfam.xfam.org/, accessed on 7 September 2023)[27]. In addition, 43 LBD protein sequences of Arabidopsis were obtained from the TAIR database (www.arabidopsis.org, accessed on 12 August 2023)[24] and used to perform a BLASTp search with an E-value threshold set at e−5. Then, SMART (http://smart.embl-heidelberg.de/, accessed on 9 September 2023)[28] and CDD (www.ncbi.nlm.nih.gov/cdd, accessed on 9 September 2023)[29] were employed to verify the presence of a LOB domain in putative LBD proteins. Subsequently, the ExPASy-ProtParam tool (https://web.expasy.org/protparam/, accessed on 10 September 2023)[30] was used to analyze the physical and chemical properties of all identified LBD proteins.

    • The presumed patterns of LBD proteins were examined using the MEME suite (https://meme-suite.org/meme/tools/meme, accessed on 10 September 2023)[31] with the following parameters: a motif number of 20, minimum width of six, maximum width of 50. CltustalW software was used for multiple sequence alignment of LBD proteins. Then, the WebLogo3 website (https://weblogo.threeplusone.com/create.cgi, accessed on 12 September 2023) was used to generate the conserved motif logos. The alignment of all LBD proteins from Arabidopsis, poplar, and Rosaceae species was constructed by a Muscle method. Following the alignment results, phylogenetic trees were created using the maximum likelihood (ML) method, and the bootstrap was set to 5,000. Using TBtools (v. 2.003) software[32], phylogenetic trees were created.

    • The chromosomal lengths and locations of LBDs were extracted from the genome database. Then, the chromosomal location figures were created by TBtools software[32]. The segment and tandem duplication events of LBDs were analyzed by McscanX with default settings[33]. The intra-species synteny relationships of nine Rosaceae genomes LBDs and the inter-species synteny relationships among Arabidopsis, poplar, and nine Rosaceae genomes were identified by MCScanX[33], and the collinearity results were visualized using TBtools (v. 2.003)[32]. The Ks, Ka, and Ka/Ks values of gene pairs were calculated using the Ka/Ks calculator in TBtools (v. 2.003)[32].

    • To further explore the biological processes involved with LBD proteins, the GO annotation of nine Rosaceae LBDs was analyzed using GO Enrichment in TBtools (v. 2.003)[32]. We extracted the 2,000-bp promoter sequences upstream of each identified LBD member and submitted them to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 19 September 2023)[34] for cis-acting element analysis. TBtools (v. 2.003) was used for visualization[32].

    • To determine the tissue-specificity of the PmLBDs, we analyzed the expression patterns of PmLBDs using RNA-seq data obtained from five distinct tissues of the mei plant (bud, root, fruit, leaf, and stem) (Accession No. GSE40162)[35]. The responses of PmLBDs to natural cold were examined using RNA-seq data from three locations (Beijing [N39°54′, E116°28′], Chifeng [N42°17′, E118°58′], and Gongzhuling [N43°42′, E124°47′], China) and phenological stages (autumn, winter, and spring). To identify the responses of PmLBDs to the regulation of flowering, RNA-seq data (accession numbers: PRJNA833165, PRJNA832606, and PRJNA832060) were downloaded from the NCBI website (www.ncbi.nlm.nih.gov, accessed on 22 September 2023)[36]. Additionally, we also examined PmLBDs expression at eight developmental stages of upright and weeping branches in the mei F1 population. The heatmaps of PmLBDs expression levels were created using TBtools (v. 2.003)[32].

      In the qRT-PCR procedure, total RNA was extracted from young stems using the RNAprep Pure Plant Plus Kit (DP441, TIANGEN). Subsequently, first-strand cDNAs were generated from 1 μg of total RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (RR047, TaKaRa, Bejing, China). To verify the accuracy of the results, nine PmLBDs were selected for qRT-PCR, and gene-specific primers used in qRT-PCR were designed by the NCBI primer tool (Supplemental Tables S1 & S2). The qRT-PCR was performed as described previously for the reaction system and conditions using the SYBR Premix Ex Taq II kit (RR820, TaKaRa) on a PikoReal real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA)[36]. The gene expression levels were determined by applying the 2−ΔΔCᴛ method, with PmPP2A serving as the internal reference gene[36].

    • Based on BLASTP and HMMER, a total of 406 LBDs were identified in the Rosaceae family. For Rosoideae, black raspberry (Rubus occidentalis), strawberry (Fragaria vesca), and Chinese rose (Rosa chinensis), 39, 34, and 39 LBDs were detected, respectively. The number of LBDs in the Amygdaloideae was comparable, with 42 in peach (Prunus persica) and 41 in the other three plants (Table 1). For Maloideae, the maximum number of LBDs was 69 in apple (Malus domestica) (Table 1). The proportion of LBDs was the highest in mei(P. mume var. tortuosa), followed by apple and hawthorn (Crataegus pinnatifida), and peach was the least (Table 1). The 406 LBD proteins of Rosaceae encoded 80 to 1,099 aa (amino acid), with molecular weights ranging from 8.90 kDa to 125.60 kDa and theoretical pI from 4.64 to 10.72. The mean hydropathicity value of just 12 proteins exceeded 0, suggesting that the majority of proteins exhibited hydrophilic properties (Supplemental Table S3). Subcellular localization prediction of all LBDs was localized in the nucleus (Supplemental Table S3).

      Table 1.  Number of LBDs in nine Rosaceae species.

      Traditional subfamilyGenus nameCommon nameSpecies nameChromosome numberGenome gene numberIdentified LBDsProportion
      of LBDs
      RosoideaeRubusBlack raspberryRubus occidentalis833,286390.12%
      FragariaStrawberryFragaria vesca728,588340.12%
      RosaChinese roseRosa chinensis739,669390.10%
      AmygdaloideaePrunusPeachPrunus persica847,089420.09%
      PrunusApricotPrunus armeniaca830,436410.13%
      PrunusMeiPrunus mume var. tortuosa826,015410.16%
      MaloideaeCrataegusHawthornCrataegus pinnatifida1740,571600.15%
      PyrusEuropean pearPyrus communis1737,445410.11%
      MalusAppleMalus domestica 'HFTH1'1744,677690.15%
    • To better analyze the evolutionary trajectory of LBD proteins in nine Rosaceae species, a maximum likelihood (ML) tree was constructed with LBDs from the Rosaceae family (406), A. thaliana (43), and P. trichocarpa (80). Based on the classification of Arabidopsis and poplar, 406 LBD proteins were divided into two major groups, Class I and Class II (Fig. 1a & Supplemental Tables S4, S5). Most proteins belong to Class I, which contained 349 (85.96%) members in nine species, while Class II had 57 (14.04%) LBD members (Fig. 1a, 1b & Supplemental Table S4). Subsequent studies revealed that Class I could be categorized into five subclasses (Class Ia-Ie), while Class II could be further separated into subclass IIa and subclass IIb. Each subclass included the LBDs of these 11 species, but there were differences in the distribution of members among different species (Fig. 1a, 1b & Supplemental Table S4). Subclass Ia had the largest number of LBDs (11) in apple, and subclass Ic contained the most members in hawthorn. Interestingly, subclass Ie had the highest number of members in most Rosaceae plants, such as strawberry, peach, mei, and apple (Fig. 1b & Supplemental Table S4). In addition, we found that the number of subclass Ia, Ic, and IIa in Rosoideae was less than that in Maloideae. In the Rosoideae and Amygdaloideae, the number of subclass IIa and IIb was consistent (Fig. 1b & Supplemental Table S4).

      Figure 1. 

      Phylogenetic tree, conserved domains, and the gene numbers of the subfamily in nine Rosaceae species. (a) ML phylogenetic tree of LBD proteins in 11 plant genomes. (b) The number of genes identified in different classes of the LBD family. (c) Analysis of three conserved domains of LBD proteins in nine Rosaceae genomes.

      The investigation of protein domain positioning and structure involved the utilization of ClustalW for carrying out multiple sequence alignment. Additionally, conserved motif logos were developed using the WebLogo3 website. Consequently, nearly all LBDs exhibited the zinc finger-like domain (CX2CX6CX3C) and GAS blocks, whereas Class II LBDs did not include the leucine zipper-like motif (LX6LX3LX6L) (Fig. 1c).

      To delve more into the functional variety and evolutionary relationship of LBDs in species belonging to the Rosaceae family, we constructed an independent phylogenetic tree for each subclass and analyzed motifs and domains within these proteins. The subclass exhibited significant variation in both the quantity and diversity of motifs (Fig. 2, Supplemental Figs S1S5). For example, subclass IIa possessed the lowest number of motifs and only nine types of motifs, while subclass Ie possessed the highest number of motifs with 15 types. Besides, the conserved motifs 1, 2, 10, and 13 were shared by each subclass. Class II did not contain motifs 4 and 6, but these only contained motifs 5 and 12. Subclass Id and Ie were the only subclasses that included motif 6, while motif 16 was exclusively found in subclass IIb. The presence of specific motifs in the LBD subclass indicated that they also had specific roles.

      Figure 2. 

      Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass Ia subfamily members.

    • To gain a deeper understanding of the evolutionary connection between LBDs in Rosaceae species, we conducted a study on the chromosome localization, collinearity analysis, and gene duplication relationship of LBDs in the nine Rosaceae genomes. Through chromosome localization, 406 LBDs in nine Rosaceae genomes were unevenly distributed across the chromosome (Fig. 3 & Supplemental Figs S6, S7). We also found that except for Maloideae, most species had LBDs distribution on each chromosome (Fig. 3, Supplemental Figs S6, S7). Specifically, no LBDs were located on chromosome 4 and 13 in pear and apple (Supplemental Fig. S7b, S7c). Chromosome 7 (Chr7) had the maximum number of LBDs in black raspberry, strawberry, pear, and apple, with 12, 9, 10, and 7, respectively (Supplemental Figs S6a, S6b, S7b & S7c). Nine and 11 LBDs were located in Chr5, which was the largest number in apricot and mei, respectively (Fig. 3b, 3c).

      Figure 3. 

      Chromosome distribution of LBDs in the Amygdaloideae. (a) Chromosome distribution of LBDs in P. persica. (b) Chromosome distribution of LBDs in P. armeniaca. (c) Chromosome distribution of LBDs in P. mume.

      In addition, collinearity analysis showed that the most segment duplication gene pairs occurred in Maloideae, such as 87 gene pairs in hawthorn, followed by Amygdaloideae, and the least in Rosoideae, such as only six in Chinese rose (Fig. 4, Supplemental Fig. S8 & Supplemental Table S6). Notably, the tandem duplication gene pairs showed significant similarity among the species of the Rosaceae family. In apple, the highest number of tandem duplication gene pairs observed was five, whereas in pear, the lowest number was two (Supplemental Fig. S3b, 3c & Supplemental Table S7). The prevalence of segment duplication genes, as opposed to tandem duplication genes, indicates that segment duplication is the primary factor responsible for the expansion of LBDs in the Rosoideae and Amygdaloideae. Subsequently, genome collinearity of LBDs among the Rosaceae family, A. thaliana, and P. trichocarpa was conducted on account of species’ evolutionary relationships. The findings indicated a significant collinearity relationship across Rosaceae plants, as depicted in Fig. 5. In Rosoideae, 46 and 42 pairs of orthologous LBDs were detected between black raspberry and strawberry and strawberry and Chinese rose, respectively (Fig. 5). A total of 53 and 49 homologous gene pairs were found in Amygdaloideae (peach vs apricot, apricot vs mei) (Fig. 5). For Malodieae, there were the most gene pairs, with 103 pairs between hawthorn and pear (Fig. 5).

      Figure 4. 

      Collinearity of segmental duplication gene pairs of LBDs in six Rosaceae species. (a) Collinearity of segmental duplication gene pairs of LBDs in R. occidentalis. (b) Collinearity of segmental duplication gene pairs of LBDs in F. vesca. (c) Collinearity of segmental duplication gene pairs of LBDs in R. chinensis. (d) Collinearity of segmental duplication gene pairs of LBDs in P. persica. (e) Collinearity of segmental duplication gene pairs of LBDs in P. armeniaca. (f) Collinearity of segmental duplication gene pairs of LBDs in P. mume. The red lines represent the segment duplication (SD) gene pairs of the LBDs.

      Figure 5. 

      Collinearity analysis of LBDs in different genomes. Colored circular rectangles denote the chromosomes of different plants. The green lines represent gene pairs with a collinear relationship. The grey lines represent other collinear gene pairs of non-LBD gene family members across genomes.

      To conduct a more in-depth examination of the rate at which LBDs have evolved in nine Rosaceae species, we computed the Ka (non-synonymous substitution) to Ks (synonymous substitution) ratio for each pair of genes. In our study, the Ks value of gene pairs was mainly distributed at 1.0 to 2.5 in black raspberry, strawberry, apricot, mei, and pear (Fig. 6a & Supplemental Data S1). The main distribution of Ks in other Rosaceae species was 2.0 to 2.5 (Fig. 6a & Supplemental Data S1). In addition, the value of Ks peaked at 2.0-2.5 in strawberry, apricot, mei, and pear, while the peak value was 1.5-2.0 in the other six plants (Fig. 6a & Supplemental Data S1). The majority of the LBD gene pairs exhibited Ka/Ks ratios below 1 (Fig. 6b & Supplemental Data S2), indicating that these genes are likely subject to purifying selection. However, it is worth mentioning that there was one gene pair in peach and two gene pairs in black raspberry with a Ka/Ks value greater than 1 (Fig. 6b & Supplemental Data S2), implying that these genes may undergo functional divergence owing to positive selection.

      Figure 6. 

      The Ks and Ka/Ks values of LBDs in nine Rosaceae genomes. (a) The distribution of Ks values among LBDs in nine Rosaceae genomes. (b) The distribution of Ka/Ks values among LBDs in nine Rosaceae genomes.

    • To further explore the biological processes involved in LBDs, we performed a gene ontology (GO) analysis of Rosaceae LBDs. According to the cellular component results, LBDs were involved in the nucleus, membrane-bounded organelle, intracellular membrane-bounded organelle, organelle and intracellular organelle (Fig. 7a). Regarding molecular function, LBDs primarily participate in protein dimerization activity and protein binding. In addition, these genes were implicated in more than 130 biological processes, including post-embryonic, plant organ, root, flower, and other developmental and morphogenetic processes, as well as phloem or xylem histogenesis, cellular response to jasmonic acid stimulus and jasmonic acid mediated signaling pathway (Fig. 7a).

      Figure 7. 

      GO and cis-elements analysis of LBDs in nine Rosaceae species. (a) GO analysis of LBDs in nine Rosaceae species. (b) The proportion of cis-elements predicted in the promoters of LBDs. (c) Numbers of the cis-elements involved in light response, hormone response, biotic and abiotic stress, development, and tissue specificity.

      To explore the potential regulatory mechanisms of LBDs, cis-acting element analysis was performed on the region 2,000 bp upstream of 406 LBDs using the PlantCARE database. The findings indicated that a total of 70,479 cis-acting elements were identified, with an average of 173 per gene (Fig. 7b & Supplemental Fig. S9). The promoter region of LBDs exhibited a widespread presence of common regulatory components, namely the CAAT-box and TATA-box, which accounted for 21.48% and 26.61% respectively. Subsequently, 20 major cis-elements were selected for further analysis (Fig. 7b). These cis-elements mainly contained: (1) light response-related elements, with an average of 12 elements per gene; (2) hormone response-related elements, such as abscisic acid, MeJA, auxin, gibberellin; (3) biotic and abiotic stress-related elements, including anaerobic induction, low-temperature, drought-inducibility, defense, and stress responsiveness; (4) development and tissue specificity related elements, such as meristem expression, wound-responsive, cell cycle regulation, circadian control, endosperm expression, seed-specific, root-specific (Fig. 7b, c & Supplemental Fig. S9). Furthermore, despite the distribution of various cis-elements throughout the promoter, the presence of similarly organized cis-acting elements on related gene promoters implies that these genes may have comparable roles (Supplemental Fig. S9). Overall, these results indicated that LBDs may play important roles as transcription factors in many biological processes and could respond to hormone response and stress.

    • To characterize the tissue-specific PmLBD gene in P. mume, the expression patterns of PmLBD family members were based on RNA-seq data. Among 41 PmLBDs, 26 PmLBDs were expressed in at least tissues (bud, root, fruit, leaf, and stem) (Fig. 8a). The PmLBDs that were considered to be tissue-specifically expressed with RPKM > 2-fold over other tissues were as follows: PmLBD3/6/13/27/29/31/34, PmLBD7/15/17/25/36/40, PmLBD8/23/24, PmLBD28 were expressed in the roots, buds, fruit and stems, respectively (Fig. 8a). The other genes were expressed in two or more tissues, among which PmLBD1 was highly expressed in all five tissues (RPKM > 40) (Fig. 8a). These findings implied that the growth and development process of tissues were regulated by these PmLBDs.

      Figure 8. 

      Expression pattern of PmLBDs in different tissues and different developmental stages of flower buds. (a) Hierarchical clustering of expression profiles of PmLBDs in different tissues (bud, fruit, leaf, root, and stem). (b) Expression profiles of PmLBDs in the flower bud during dormancy release.

      To look into the potential role of PmLBDs in the regulation of blooming, particularly in the process of floral bud break, we assessed the expression levels of PmLBDs at four different stages of floral bud dormancy release in P. mume. As shown in Fig. 8b & Supplemental Fig. S10, PmLBD2 exhibited a continuous upregulation with floral bud exit dormancy, while PmLBD12/35 showed a downregulation trend. PmLBD19 expression was suppressed in the endodormancy process, increased during ecological dormancy, and decreased sharply at bud flush, while PmLBD6 was up-regulated during endodormancy and decreased after ecological dormancy. These results demonstrate that these PmLBDs function in floral bud dormancy release.

      To examine how PmLBDs react to cold stress in mei, we analyzed the expression patterns in the stem at three different locations throughout three time periods. The expression level of PmLBDs varied greatly at different geographic locations and in different periods (Fig. 9 & Supplemental Fig. S11). For example, PmLBD1/6/13/17 showed large expression levels under all three locations at the same time. In addition, PmLBD13 exhibited an initial downregulation followed by an upregulation trend at three test sites, while PmLBD26 showed an opposite trend (Fig. 9 & Supplemental Fig. S11). Notably, some genes showed inconsistent expression at three test sites. For example, the expression of PmLBD1 exhibited down-regulation in winter and up-regulation in spring in Beijing, a continuous down-regulation trend in Chifeng, and upregulation followed by downregulation in Gongzhuling (Fig. 9a). PmLBD19 showed a continuous up-regulation in Beijing, down-regulation in winter, and up-regulation in spring in Chifeng, and an increase followed by a downregulation in Gongzhuling (Fig. 9b). These results suggest that PmLBDs were involved in the response to cold stress in P. mume.

      Figure 9. 

      Expression pattern of PmLBDs in different locations and seasons. (a) Hierarchical clustering of expression profiles of PmLBDs in different locations. (b) Hierarchical clustering of expression profiles of PmLBDs in different seasons. BJ, Beijing; CF, Chifeng; GZL, Gongzhuling.

      We also examined the expression of PmLBDs at eight developmental stages of upright and weeping branches in the mei F1 population. The PmLBDs showed a large variation in expression patterns during branch development (Fig. 10). The expression of PmLBD5 showed a continuous upregulation at eight developmental stages, and PmLBD28/30/40 exhibited an up-regulation followed by a decrease. Notably, PmLBD6 was consistently higher in weeping branches than in upright branches, while PmLBD20 showed an opposite trend (Fig. 10). To verify the accuracy of the transcript levels of PmLBDs in transcriptome data, nine candidate genes were selected based on the subfamily classification and differential gene clustering. Their expression level in upright and weeping branches was investigated using qRT-PCR with PmPP2A as a reference gene. Finally, RNA-seq data were consistent with the qRT-PCR results (Fig. 11).

      Figure 10. 

      Expression pattern of PmLBDs in upright and weeping branches. U1−U8, eight developmental stages of upright branches in the mei F1 population; W1−W8, eight developmental stages of weeping branches in the mei F1 population.

      Figure 11. 

      qRT-PCR analysis of nine PmLBDs in upright and weeping branches. U1−U8, eight developmental stages of upright branches in the mei F1 population; W1−W8, eight developmental stages of weeping branches in the mei F1 population. The relative quantification method (2−ΔΔCᴛ) was used to evaluate quantitative variation. Error bars represent standard error for three replicates.

    • The LBDs, which are exclusive to plants, play a crucial role in regulating a wide range of biological activities such as plant secondary metabolism, growth and development, and response to different types of stress[11,13,22]. Due to their crucial function in plant development, LBDs have been extensively researched in several plant species. Rosaceae is one of the important plant families, however, comparative studies on LBDs in Rosaceae remain unknown. In this study, 42 AtLBDs from Arabidopsis were utilized to identify LBD proteins in nine representative Rosaceae plants, with the number of LBDs ranging from 34 to 69, independent of genome size. The number of LBDs was similar in most selected plants, but was far greater in hawthorn and apple with 60 and 69 LBDs, respectively. The presence of a large number of LBDs in hawthorn and apple may be related to the widespread occurrence of duplication events in their genomes[37,38].

      Four hundred and six LBD proteins could be categorized into two classes: Class I (349, 85.96%) and Class II (57, 14.04%), and LBDs in Class I was significantly higher than in Class II among all of the selected Rosaceae plants, which is consistent with previous results[11]. Similar to the previous phylogenetic tree results[11,39], those proteins were divided into seven major subclasses, and the LBDs of these 12 species were distributed in each subclass. The classification was further supported by gene motif analysis and structural domains, indicating that genes in the same subclass usually have similar biological functions. Recent studies have reported that root-type-specific regulation by subclass IB LBDs is deeply conserved[22]. For example, Solyc09g066270, a subclass IB LBD, could specifically regulate the earliest stage of root initiation[22]. Therefore, we hypothesize that subclass IB LBDs may play a deeply conservative role in lateral root initiation and provide reference genes for difficult-to-root plants in Rosaceae, especially in the Prunus genus.

      Gene duplication is a crucial factor in the process of evolution and the growth of gene families. Segmental and tandem duplication are the primary mechanisms for gene family growth[40]. A total of 242 pairs of LBD duplication genes were discovered in the nine Rosaceae genomes under analysis. Out of these, 215 pairs were recognized as segment duplication genes, while only 27 pairs were classified as tandem duplication. This finding demonstrates that segmental duplication events play a crucial role in driving the proliferation of the LBD family in the Rosoideae and Amygdaloideae. In addition, synteny analysis revealed a great collinearity relationship among Rosaceae plants, especially those in Malodieae, which proved that the LBDs are relatively conserved in Rosaceae, suggesting that the functions of these homologous genes may be consistent. The Ka/Ks ratio was used to measure the selection pressure experienced by the gene pairs. Previous studies have shown that LBDs proceed with a purifying selection in moso bamboo (Phyllostachys edulis) and ginkgo (Ginkgo biloba)[41,42]. Similarly, almost all LBDs in selected Rosaceae in this study have undergone purifying selection. The presence of a few LBDs may undergo functional divergence owing to positive selection in black raspberry and peach, indicating that functional differentiation of these genes may occur, which is worthy of further research.

      GO analysis and promoter cis-acting element prediction can determine the possible functions of LBDs. AtLBD3 and AtLBD4 have been reported to activate secondary growth through cytokinin signaling[43]. AtLBD16/17/18/29 plays key roles in plant regeneration programs mediated by the auxin signals[12]. Our study revealed that the LBD promoters contain many kinds of hormone response-related elements (abscisic acid, MeJA, auxin, and gibberellin). Thus, we postulated that LBDs might play a role in plant development through their reaction to hormones. Furthermore, the LBD promoters were shown to contain components associated with light, low temperature, drought, defense and stress responses, meristematic organization, and tissue specialization. These findings indicate that LBDs have a significant impact on various biological processes and may be involved in hormone response and stress. This is consistent with the results of the PmLBDs expression analysis under low temperature and tree architecture in this study.

      The LBDs have been extensively reported to play a crucial role in controlling the development of many plant parts, including roots, flowers, leaves, and stems. This finding is congruent with the results obtained from the GO enrichment study. GO annotations of 470 LBDs contained a variety of plant organ development and formation, including post-embryonic, plant organ, root, and flower. In Arabidopsis, AtLBD13[44], AtLBD16[45], and AtLBD33[46] were shown to play key roles in controlling lateral root development. PmLBD3 with AtLBD13, PmLBD27 with AtLBD33, and PmLBD31 with AtLBD16 were respectively in the same subclades. Notably, the three genes were specifically expressed in the root. Therefore, we hypothesized that PmLBD3/27/31 may be involved in lateral root development.

      Previous studies showed that LBDs respond positively to various abiotic stresses. For example, in Ginkgo, GbLBD31, a pleiotropic regulator, was significantly expressed under drought and cold stress[42]. In banana, MaLBD5 may be associated with MeJA-induced cold tolerance and activated jasmonate biosynthesis gene[23]. In our study, the response of PmLBDs to cold stress was revealed at three sites for three periods by analyzing previous transcriptome data. Our investigation revealed significant variation in the expression of PmLBDs across different geographic locations and periods. Additionally, we observed inconsistent expression of certain genes among the three investigated loci. Notably, four differentially expressed genes (DEGs) were predicted to contain low-temperature response elements, eight DEGs contained MeJA response elements, and three DEGs contained both low-temperature and MeJA response elements, indicating that these genes potentially have a role in the development of cold tolerance in mei driven by MeJA and provide potential candidate genes for future research on cold tolerant molecular breeding in mei.

      It is known that flowering transition is controlled by the gene regulatory network. In rice, OsLBD37 and OsLBD38 were found to delay flowering by down-regulating Hd3a and RFT1 expression[47]. In transgenic Arabidopsis, CsLBD37 overexpression affects nitrogen-responsive gene expression and nitrate content, which may regulate early flowering in plants through nitrogen signaling[48]. The study observed significant alterations in the expression levels of many LBDs, suggesting their potential involvement in the regulation of blooming.

      Prior research has demonstrated that the abnormal growth of phloem, namely the lack of phloem fibers, had a significant role in the development of weeping traits in mei[49]. PtaLBD1 and PtaLBD4 regulate the development of the secondary phloem by inhibiting the expression of identity genes in the meristem[11]. In overexpression poplar, EgLBD29 controls secondary growth especially the development of phloem fiber[11]. This suggests that LBDs may regulate secondary growth in plants, especially phloem fiber. In this study, PmLBD6 was consistently higher in weeping branches than in upright branches, while PmLBD20 showed an opposite trend. Notably, in mei, PmLBD30, the ortholog of EgLBD29, exhibited an up-regulation followed by a decrease, which is hypothesized to possibly play a role in branch development and can be studied as a candidate gene for the formation of a weeping trait in mei.

    • In Rosaceae, 39, 34, 39, 42, 41, 41, 41, 60, 41, and 69 LBDs were identified in black raspberry, strawberry, Chinese rose, peach, apricot, mei, pear, hawthorn, and apple, respectively. Among them, the LBDs were classified into seven major subclasses. The primary factor responsible for the amplification of LBDs in Rosaceae plants was the duplication of segments. Phylogenetic tree and RNA-seq data showed that PmLBD27/31 were tissue-specifically expressed in the roots. Transcription sequencing data from three locations for three periods indicated that PmLBD17/19/41 were induced by low temperature and they all contained both low-temperature and MeJA response elements. Moreover, PmLBD30 exhibited an up-regulation followed by a decrease in the developmental stages of branches. In summary, our studies offer novel perspectives on the evolutionary connection between the LBD family in Rosaceae and the role of LBDs in mei.

    • The authors confirm contribution to the paper as follows: study conception and design: Liu W, Zheng T; conducted experiments and material collection: Zheng T, Liu W, Guo X; data analysis: Liu W, Guo X, Zheng T; conducted fieldwork and material maintenance: Liu W, Guo X, Li X, Wang J, Cheng T, Zhang Q; modified the language modification: Ahmad S; draft the manuscript: Liu W, Guo X, Zheng T; manuscript revision and finalization: Zheng T, Cheng T. 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.

      • The research was supported by the National Natural Science Foundation of China (No. 32371947), the Beijing High-Precision Discipline Project, Discipline of Ecological Environment of Urban and Rural Human Settlements, and the Special Fund for Beijing Common Construction Project.

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

      • # Authors contributed equally: Weichao Liu, Xiaoyu Guo

      • Supplemental Tables S1 Primers used in this study.
      • Supplemental Tables S2 The reference sequences used for gene-specific primers in Prunus mume.
      • Supplemental Tables S3 The physicochemical parameters of all LBD proteins in ten Rosaceae genomes.
      • Supplemental Tables S4 The number of LBDs of eleven species in each subclass.
      • Supplemental Tables S5 Classification of LBD in each subclass.
      • Supplemental Tables S6 Segmental duplication events of nine Rosaceae species.
      • Supplemental Tables S7 Tandem duplication events of nine Rosaceae species.
      • Supplemental Date S1 Ks values of nine Rosaceae species
      • Supplemental Date S2 ka/Ks values of nine Rosaceae species
      • Supplemental Fig. S1 Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass Ib subfamily members.
      • Supplemental Fig. S2 Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass Ic subfamily members.
      • Supplemental Fig. S3 Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass Id subfamily members.
      • Supplemental Fig. S4 Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass Ie subfamily members.
      • Supplemental Fig. S5 Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass IIa and IIb subfamily members. (A) Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass IIa subfamily members. (B) Phylogenetic evolutionary tree, motifs distributions, and domains of the subclass IIb subfamily members.
      • Supplemental Fig. S6 Chromosome distribution of LBDs in the Rosoideae. (A) Chromosome distribution of LBDs in R. occidentalis. (B) Chromosome distribution of LBDs in F. vesca. (C) Chromosome distribution of LBDs in R. chinensis.
      • Supplemental Fig. S7 Chromosome distribution of LBDs in the Maloideae. (A) Chromosome distribution of LBDs in C. pinnatifida. (B) Chromosome distribution of LBDs in P. communis. (C) Chromosome distribution of LBDs in M.domestica.
      • Supplemental Fig. S8 Collinearity of segmental duplication gene pairs of LBDs in the Maloideae. (A) Collinearity of segmental duplication gene pairs of LBDs in C.pinnatifida. (B) Collinearity of segmental duplication gene pairs of LBDs in P.communis. (C) Collinearity of segmental duplication gene pairs of LBDs in M.domestica. The red lines represent the segment duplication (SD) gene pairs of the LBDs.
      • Supplemental Fig. S9 The distribution of the main 20 cis-elements in LBD gene promoters.
      • Supplemental Fig. S10 Expression pattern of PmLBDs in different developmental stages of flower buds.
      • Supplemental Fig. S11 Expression pattern of PmLBDs in different locations and seasons. BJ, Beijing; CF, Chifeng; GZL, Gongzhuling.
      • 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 (11)  Table (1) References (49)
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    Liu W, Guo X, Zheng T, Li X, Ahmad S, et al. 2024. Genome-wide identification and characterization of the Lateral Organ Boundaries Domain (LBD) gene family in nine Rosaceae species and expression pattern in Prunus mume. Ornamental Plant Research 4: e007 doi: 10.48130/opr-0024-0005
    Liu W, Guo X, Zheng T, Li X, Ahmad S, et al. 2024. Genome-wide identification and characterization of the Lateral Organ Boundaries Domain (LBD) gene family in nine Rosaceae species and expression pattern in Prunus mume. Ornamental Plant Research 4: e007 doi: 10.48130/opr-0024-0005

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