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Ammonium and nitrate impact petal color traits and amino acid profiles differently in Petunia

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  • Nitrogen (N) is vital for ornamental plants to fulfill fundamental cellular functions. Effects of inorganic N, including nitrate and ammonium, on ornamental traits at the biochemical level are less reported. Given that the continuously increased concentration of CO2 in the atmosphere has greater negative effects on nitrate reduction than ammonium utilization in C3 plants, it is vital for us to dissect various effects of different inorganic N forms on flower development, which will lead to a better understanding of the metabolic regulation of ornamental traits. In this study, Petunia × hybrida cv 'Mitchell Diploid' (MD) plants at 6−8-leaf stage were treated hydroponically with the same and regular amount of inorganic N for one month but in three different forms, 1 mM NH4NO3 as the control (NH4NO3), 2 mM NH4Cl alone (NH4-N) and 2 mM NaNO3 alone (NO3-N). Results indicated that relative to the control, effects of NH4-N and NO3-N treatment alone on plant growth, N utilization and distribution, petal color traits and petal amino acid profiles were different by varying degrees. We have confirmed that NH4-N could specifically increase 15N distribution to floral tissues, change the amino acid profiles in petals which in turn bring changes to petal colors. Results with four commercially available Petunia cultivars with different flower colors also validated our findings. This study has been designed to specifically investigate effects of ammonium or nitrate as the sole source of inorganic N on the developmental and chemical features of floral tissues in Petunia.
  • Huangqin tea (HQT, Fig. 1), also called Huangjin tea in Chinese, has a long history of consumption in China[1]. It is a healthy herbal tea crafted from the aerial parts of Scutellaria baicalensis Georgi, S. scordifolia Fisch, S. amoena C. H. Wrigh, and S. viscidula Bung[24]. Unlike traditional tea made from Camellia sinensis (L.) O. Kuntze, HQT lacks stimulating components and is renowned for clearing heat and dry dampness, eliminating toxins, promoting digestion, and soothing fire[1,5]. It can be enjoyed both as a hot beverage and as a cold drink, and modern research suggests that HQT exhibits several pharmacological properties, such as anti-inflammatory, chemopreventive effects of colorectal cancer[6], anti-aging[7], cardiovascular protection, hypoglycemic effect, hypolipidemic effect, anti-tumor, anti-bacterial, anti-influenza virus, and enhance human resistance[812]. The predominant compounds found in HQT are flavonoids and essential oils and its potential health benefits are likely attributed to the presence of distinctive flavonoids, including isocarthamidin-7-O-β-D-glucuronide, carthamidin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucopyranside, chrysin-7-O-β-D-glucuronide, scutellarin, baicalin, wogonoside, and chrysin[1318].

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  • Supplemental Table S1 Concentrations of 14 free amino acids in Petunia petals at three developmental stages measured via GC-MS for each treatment (SE, n=4). Different letters in each row indicate significant difference between treatments using one-way ANOVA test in SPSS software at P < 0.05.
    Supplemental Table S2 CIELab coordinates including perceptual lightness (L*), redness/greenness (a*), yellowness/blueness (b*), chroma (C*), and hue angle (h°) in petals of four petunia cultivars measured for each treatment via a portable colorimeter. Detailed data of four biological replicates for each treatment were presented here.
    Supplemental Fig. S1 Effects of ammonium-nitrate (NH4NO3), ammonium (NH4-N), and nitrate (NO3-N) on petal color traits in four commercially available petunia cultivars. Changes in CIELab coordinates including L*, a*, b*, C*, and h° in white- (a), pink- (b), dark pink- (c) and red-flower cultivar (d) were plotted in column figures (SE, n=4; scale bar = 1cm). Different letters above the bars in each column indicate significant difference between treatments using one-way ANOVA test in SPSS software at P < 0.05.
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  • Cite this article

    Guo H, Wang G, Zhang P, Wang G, Bao Z, et al. 2022. Ammonium and nitrate impact petal color traits and amino acid profiles differently in Petunia. Ornamental Plant Research 2:14 doi: 10.48130/OPR-2022-0014
    Guo H, Wang G, Zhang P, Wang G, Bao Z, et al. 2022. Ammonium and nitrate impact petal color traits and amino acid profiles differently in Petunia. Ornamental Plant Research 2:14 doi: 10.48130/OPR-2022-0014

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Ammonium and nitrate impact petal color traits and amino acid profiles differently in Petunia

Ornamental Plant Research  2 Article number: 14  (2022)  |  Cite this article

Abstract: Nitrogen (N) is vital for ornamental plants to fulfill fundamental cellular functions. Effects of inorganic N, including nitrate and ammonium, on ornamental traits at the biochemical level are less reported. Given that the continuously increased concentration of CO2 in the atmosphere has greater negative effects on nitrate reduction than ammonium utilization in C3 plants, it is vital for us to dissect various effects of different inorganic N forms on flower development, which will lead to a better understanding of the metabolic regulation of ornamental traits. In this study, Petunia × hybrida cv 'Mitchell Diploid' (MD) plants at 6−8-leaf stage were treated hydroponically with the same and regular amount of inorganic N for one month but in three different forms, 1 mM NH4NO3 as the control (NH4NO3), 2 mM NH4Cl alone (NH4-N) and 2 mM NaNO3 alone (NO3-N). Results indicated that relative to the control, effects of NH4-N and NO3-N treatment alone on plant growth, N utilization and distribution, petal color traits and petal amino acid profiles were different by varying degrees. We have confirmed that NH4-N could specifically increase 15N distribution to floral tissues, change the amino acid profiles in petals which in turn bring changes to petal colors. Results with four commercially available Petunia cultivars with different flower colors also validated our findings. This study has been designed to specifically investigate effects of ammonium or nitrate as the sole source of inorganic N on the developmental and chemical features of floral tissues in Petunia.

    • Production and care of ornamental plants usually receive less attention relative to other horticultural crops and therefore may suffer more from suboptimal growth conditions. It is vital for us to understand the development of ornamental plants at the biochemical level in response to environmental cues, which will lead to a healthier cultivation systems.

      Growth of plants is inseparable from the supply of nitrogen (N)[1]. Nitrogen plays key roles in the formation of cells, tissues and organs as the primary constituent of proteins, nucleic acids, nucleoside phosphates, phospholipids, and more importantly, secondary metabolites[2]. Both inorganic and organic forms of N exist in plants and soils with ammonium and nitrate as the most important inorganic N for growth and development. Generally, ornamental plants prefer nitrate over ammonium[3]. Recently, several studies suggest that with the rise of atmospheric CO2 concentrations, nitrate reduction in C3 plants such as Arabidopsis and wheat is inhibited whereas ammonium utilization is unaffected[4]. This is problematic as this reduction in nitrate assimilation can lead to the decrease of N use efficiency and the accumulation of ammonium. And the latter will cause ammonium toxicity in plants via acidic stress[5] and impair shoot growth significantly which also affects the production of ornamental plants. Therefore, different forms of inorganic N should be assessed independently in horticultural crops including ornamental plants.

      Plant roots can take up both ammonium and nitrate from soil[6]. The absorption of inorganic N is an active process that is controlled by plasma membrane-localized transporters in the root[7]. There are two nitrate uptake transporter families in higher plants, namely low-affinity transporter (LATS) NRT1/PTR and high-affinity transporter (HATS) NRT2[8,9], while ammonium uptake is mediated by the high affinity transporters of the AMT/MEP/Rh (AMT) subfamily[7]. In cells, the reduction of nitrate to ammonium requires two consecutive reactions. Firstly, NO3 is reduced to nitrite in the cytoplasm by nitrate reductase (NR)[10]. Secondly, nitrite is reduced by nitrite reductase (NiR) to form ammonium in the chloroplast[11]. Ammonium is then incorporated into organic molecules via the glutamine synthase (GS)-glutamate synthase (GOGAT) pathway in leaves or roots[1214]. Sequentially a variety of amino acids are synthesized in both roots and shoots and participate in biosynthesis of diverse metabolites. On the other hand, cellular ammonium can also come directly from photorespiration and the cycling of amino acids. Nitrate assimilation and ammonium assimilation can both be regulated at the transcriptional and posttranslational level and is a core metabolic network in plant carbon-nitrogen interactions[15].

      Generally, the assimilation of ammonium is far more energy efficient compared to nitrate assimilation[16]. However, nitrate is more available for plants due to its higher mobility[17]. Also, soil aeration status and soil pH value have effects on the availability of inorganic N. In well aerated agricultural soils, nitrate is the principal inorganic N source, whereas in undisturbed soils, ammonium is dominant. Plants grown in acidic soils take up more nitrate, while in soils with neutral pH values, they prefer taking up more ammonium. Meanwhile, different plant species will preferentially select specific N sources, thereby forming different N utilization and distribution mechanisms, which are mainly related to the long-term adaptation of plants to N environments.

      The floral organ is one of the main nitrogen pools and its formation requires the regulation of multiple metabolic pathways. However, roles of N metabolism in flower development and function fulfillment are still poorly understood. Amino acid metabolism, a part of plant N metabolism, is of great significance to the development and function of floral organs[18]. In addition, amino acids are also important precursors for the synthesis of secondary metabolites, such as phenolics, alkaloids and other nitrogen-containing secondary substances. Studies have shown that the expression of the gene encoding 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in Petunia was associated with petal specificity, and the expression level was significantly increased during flower opening[19]. EPSP synthase is also a key enzyme in the shikimate pathway and is essential for the production of aromatic compounds that provide substances such as phenylpropanoid and indoleacetic acid for petal coloration and development[19,20]. Studies have shown that inhibition of EPSP synthase leads to a decrease in the level of aromatic amino acids, which in turn inhibits protein synthesis[21,22]. So far, besides studies related to amino acid transporters in various floral tissues[23,24], only a few studies have focused on amino acid metabolism in floral tissue[25]. The de novo synthesis of proline has been reported in floral tissues such as pollen grains[18]. Efforts should be made to dissect different forms of inorganic N on floral nitrogen metabolism, which will lead to a better understanding of the metabolic features in floral tissues.

      In this study, we treated Petunia plants with three forms of inorganic N at the same concentration of total N including1 mM NH4NO3 (the control), 2 mM NH4Cl (NH4-N) and 2 mM NaNO3 (NO3-N) from the vegetative to the reproductive stage. Plant growth, 15N utilization and distribution in different tissues, petal color traits and petal amino acid profiles were thoroughly analyzed to compare effects of different inorganic N on floral development and establish unique roles of ammonium in floral tissues. Results are discussed in the context of establishing roles of different forms of inorganic N in the formation of ornamental traits including flower development and petal pigmentation.

    • Petunia × hybrida cv 'Mitchell Diploid' (MD) plants were used in this study. After approaching their 6−8 leaf stage grown in culture medium (Pindstrup Sphagnum peat moss: perlite: vermiculite = 3:1:1) under 16/8-h day/night cycles, temperature of 22/18 °C, light intensity of 150 μmol m−2 s−1, and 50% relative humidity, MD plants were then hydroponically cultured with the same and regular amount of inorganic N for one month but in three different forms, 1 mM NH4NO3 as the control (NH4NO3), 2 mM NH4Cl alone (NH4-N) and 2 mM NaNO3 alone (NO3-N). This experimental set-up had been tested to make sure that after one month of treatment, MD plants could reach their reproductive stage and produce flowers for sampling. The total N content at 2 mM was chosen because it was the regular amount of N used in nutrient solution. Other than inorganic N, the nutrient solution was prepared as the following formula: 0.67 mM NaH2PO4, 0.60 mM MgC12·6H2O, 0.30mM K2SO4, and 0.30 mM CaCl2·2H2O. The pH value of nutrient solution was adjusted to 5.8. During the treatment, nutrient solutions were replaced every 10 d and clean water was supplemented as needed.

    • After 30 d of hydroponic treatment with different forms of inorganic N, the plant height, branch number, flower number, leaf area, corolla area, and the fresh and dry weight of both above- and under-ground tissues in MD plants were measured. Plant height, the natural height of the main stem, was analyzed with a tape measure. Stem thickness was the width of the main stem near the root of the plant, measured with a vernier caliper. Considering that the Petunia plant was short, the number of branches longer than 5 cm was counted. Petals and leaves were randomly collected from plants and measured for their area via Image J software (https://imagej.nih.gov/ij). Tissue weights were determined using ME820 balance. A total of 3−4 biological replicates were analyzed for each parameter.

    • In the first and second weeks of hydroponic treatment, 0.3% 15N-urea (Shanghai Research Institute, Shanghai, China), 15N natural abundance is 10.16%; Natural 15N abundance is 0.365%) solution was brushed on the same leaves. At the end of treatment, we collected roots, stems, leaves and flowers (three biological replicates) and dried them in ovens at 60 °C for 2 d to obtain the dry weight via a ME820 balance. The samples were ground with a mortar into fine powder and sieved with a 100-mesh sieve. Total N content in various tissues was measured via a Kjeldahl nitrogen determination apparatus. 15N abundance in a specific tissue (%) was determined by DELTA V Advantage isotope mass spectrometer (Thermo Fisher, MAT-251). The calculations of 15N absorption, 15N utilization rate and 15N distribution rate in each tissue were carried out according to the following equations:

      Ndff (%) = (15N abundance in a specific tissue − Natural 15N abundance) / (15N abundance in 15N urea − Natural 15N abundance) × 100;

      15N absorption (mg plant−1) = Ndff × Total N in a specific tissue;

      15N utilization rate (%) = (Ndff × Total N in a specific tissue) / Total content of 15N applied to plants × 100;

      15N distribution rate (%) = (15N absorption of a specific tissue / Total 15N absorption of 4 tissues) × 100.

      In the equations, Ndff (%) is the percentage of N derived from 15N urea; total content of 15N applied to plants was 0.4893 g according to the amount of 15N urea used inthe label experiment.

    • Petal tissues were collected at the early, middle and mature stages during the development of MD flowers and quenched with liquid nitrogen. Petal tissues (~100 mg in FW) were ground into a fine powder in liquid nitrogen and then extracted with 1.5 ml methanol/chloroform/water (4 °C). Norvaline was added to extracts as the internal standard. The extract was filtered with a 0.45 μm cellulose acetate filter and by aspiration with N2. Amino acid content was determined according to the LC-MS/MS method[26] with slight modifications. After re-dissolution with ddH2O, samples were injected into a Thermo Scientific TSQ Quantum Access MAX Triple Quadrupole Mass Spectrometer coupled with a Thermo Fisher Hypersil GOLDTM C18 column (product# 25002-102130).

    • CIELab coordinates including perceptual lightness (L*), redness/greenness (a*), yellowness/blueness (b*), chroma (C*), and hue angle () of Petunia petals were measured for each treatment via a portable colorimeter (CR-10). Four biological replicates were analyzed.

    • Data were recorded and analyzed in Excel, and statistical analyses were performed using Data Processing System (DPS) Software[27]. Duncan's Multiple Range Test was performed in DPS software to determine the statistical difference with P value less than 0.05 between treatments. Clustering analysis, correlation analysis and principal component analysis were performed in R Studio Version 1.1.456 (www.rstudio.com) with R version 3.5.2 using gplots and pheatmap (https://cran.r-project.org/web/packages/pheatmap/) packages, and factoextra (http://www.cran.r-project.org/web/packages/factoextra/) packages, respectively.

    • In this study, MD plants were treated hydroponically with the same level of inorganic N for one month but in three different forms, 1 mM NH4NO3 as the control (NH4NO3), 2 mM NH4Cl alone (NH4-N) and 2 mM NaNO3 alone (NO3-N). The concentration of total N for each treatment was set up at 2 mM as this is the regular level of N used in nutrition solutions. After one month of treatment, MD plants approached their reproductive stages and could produce a couple of flowers (Fig. 1a). We measured the total content of N in above- and underground tissues and confirmed that Petunia plants treated with NH4NO3, NH4-N and NO3-N had very similar content of total N, which were 4.0 ± 0.8, 4.0 ± 0.5 and 4.4 ± 0.2 g plant−1, respectively (Fig. 1a). Relative to the NH4NO3, NH4-N treatment only enhanced the dry weight (DW) of above-ground tissues whereas NO3-N treatment significantly increased the fresh weight (FW) and DW of above- and under-ground tissues (Fig. 1b, c). NH4-N and NO3-N treatments did not affect plant height (Fig. 1d) but increased the number of branches by 36.8% and 26.3%, respectively (Fig. 1e). NH4-N and NO3-N also significantly increased the number of flowers by 45.7% and 32.6%, respectively (Fig. 1f). However, NH4-N and NO3-N treatments greatly reduced the area of leaf and corolla (Fig. 1g, j) with NH4-N had more negative effects on leaf area than NO3-N treatment.

      Figure 1. 

      Effects of ammonium-nitrate (NH4NO3), ammonium (NH4-N), and nitrate (NO3-N) on Petunia plants. (a) Petunia plants (MD) after treatment (scale bar = 5 cm). (b), (c) Fresh weight (FW) and dry weight (DW) of above- and under-ground tissues (SE, n = 4). (d)−(f) Plant height, number of branches and number of flowers measured for each treatment (SE, n = 4). (g), (h) Close-up view of the representative, fully expanded leaves and petals of fully-opened flowers of each treatment (scale bar = 1 cm). (i), (j) Total leaf area and corolla area measured for each treatment (SE, n = 4). Different letters above the bars indicate significant difference between treatments using one-way ANOVA test in SPSS software at P < 0.05.

    • In order to determine the effects of different N forms on uptake and distribution of N in Petunia plants, we performed a labeling experiment with 15N-urea. At first, N content in each tissue was measured and the results indicated that leaves had the highest content of N relative to roots, stems and flowers. Different forms of N did not affect N conent on the level of individual tissues (Fig. 2a). The absorption, ulitization rate and distribution rate of 15N in each tissue were then calculated based on formulas described in the Materials and Methods (Fig. 2bd). Realtive to NH4NO3 treatment, NO3-N did not have effects on the tissue-level 15N absorption, utilization and distribution. In both NH4NO3 and NO3-N treatments, leaf-absorption of 15N was the highest among all tissues and roots, stems and flowers absorbed and utilized similar amounts of 15N. However, for the treatment of NH4-N, flower-absorption of 15N was significantly higher than that of roots. Therefore, in NH4-N, both 15N utilization and 15N distribution rate in flowers were significantly higher than those in roots. More importantly, among the three treatments, NH4-N had the highest 15N distribution rate in flowers.

      Figure 2. 

      Effects of ammonium-nitrate (NH4NO3), ammonium (NH4-N), and nitrate (NO3-N) on the tissue-level distribution of N within Petunia plants. (a) Nitrogen content , (b) 15N absorption content, (c) 15N utilization rate, and (d) 15N distribution rate in roots, stems, leaves and flowers measured for each treatment (SE, n = 3). Different letters above the bars in each tissue indicate significant difference between treatments using one-way ANOVA test in SPSS software at P < 0.05.

    • Considering that NH4-N dramatically changed 15N distribution into floral tissues compared to other forms of N, we measured the color traits of Petunia plants for each treatment via a portable colorimeter. Changes in CIELab coordinates including perceptual lightness (L*), redness/greenness (a*), yellowness/blueness (b*), chroma (C*), and hue angle () of Petunia petals are presented in Fig. 3. Relative to NH4NO3 treatment, both NH4-N and NO3-N treatment decreased L* and and increased b* and C* (Fig. 3a). However, there was no significant difference between NH4-N and NO3-N treatments in terms of their effects on color traits of white petals. The principal component analysis (PCA) of color traits further confirmed our findings (Fig. 3b).

      Figure 3. 

      Effects of ammonium-nitrate (NH4NO3), ammonium (NH4-N), and nitrate (NO3-N) on petal color traits. (a) CIELab coordinates including perceptual lightness (L*), redness/greenness (a*), yellowness/blueness (b*), chroma (C*), and hue angle () of Petunia petals measured for each treatment via a portable colorimeter (SE, n = 4). Colors (green to purple) and transparency of arrows toward each parameter indicate their contributions to the principal components. Different letters above the bars in each column indicate significant difference between treatments using one-way ANOVA test in SPSS software at P < 0.05. (b) The principal component analysis (PCA) of color traits in three treatments.

    • Most pigments in Petunia petals (except yellow) are derived from flavonoid and anthocyanins and amino acids were their direct precursors. We then analyzed the effects of different N forms on the petal amino acid profile in Petunia plants at three developmental stages (Fig. 4a). Via LC-MS/MS, we had identified a total of 14 amino acids in petals including four glutamate (Glu) family amino acids, five aspartate (Asp) family amino acids, three aromatic amino acids and serine and leucine (Supplemental Table S1). We first compared effects of different N forms on total content of glutamate family, aspartate family and aromatic amino acids (Fig. 4bd). Under treatment of NH4NO3, amino acid profiles displayed clear patterns of changes with the development of flowers. Content of Glu family and aromatic amino acids increased from S1 to S3 stage whereas Asp family decreased in NH4NO3 treated petals. NO3-N treated plants lost this developmental pattern with similar content of amino acids accumulated in petals throughout the three stages. NH4-N treated plants accumulated slightly more amino acids that NO3-N treated plants. More importantly, Glu family amino acids in NH4-N treated plants showed similar developmental patterns to those treated with NH4NO3, suggesting that NH4-N treatment has more positive effects on the petal amino acid profile than NO3-N treatment.

      Figure 4. 

      Effects of ammonium-nitrate (NH4NO3), ammonium (NH4-N), and nitrate (NO3-N) on petal amino acid profiles in Petunia plants. Petal tissues were collected at S1, S2 and S3 stages indicated in (a) (scale bar = 1 cm) and total concentrations of glutamate family amino acids, aspartate amino acids and aromatic amino acids (b)−(d) (SE, n = 4). Different letters above the bars indicate significant difference between treatments using one-way ANOVA test in SPSS software at P < 0.05. Concentrations (nmol g FW1) of individual amino acids are reported Supplemental Table S1.

      We then presented changes in individual amino acid content in detail (Fig. 5). As mentioned above, Glu family amino acids including Glu, glutamine (Gln), arginine (Arg) and proline (Pro) showed very similar trends in changes of their content in both NH4NO3 and NH4-N treatments but not in NO3-N treatment (Fig. 5a). Different forms of N did not affect the developmental changes of the content of threonine (Thr), isoleucine (Ile) and lysine (Lys) in NH4-N and NO3-N treatments relative to NH4NO3 (Fig. 5b). However, content of two major Asp family amino acids, Asp and asparagine (Asn), were significantly influenced by treatments of NH4-N and NO3-N mainly at the S1 stage of flower development. Aromatic amino acids displayed significant changes under various N treatments. Content of tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe) in NH4NO3 treated petals increased with flower development except at the S3 stage, content of Phe decreased (Fig. 5c). NH4-N and NO3-N treatments did not change content of Tyr, Trp and Phe significantly from the S1 to the S3 stage. To be noted, NH4-N petals did have slightly higher content of Tyr than those treated with NO3-N. Content of serine (Ser) did not significantly change with various treatments and content of leucine (Leu) in NO3-N was lower than those in the other two treatments.

      Figure 5. 

      Effects of ammonium-nitrate (NH4NO3), ammonium (NH4-N), and nitrate (NO3-N) on relative abundance of amino acids in Petunia petals. (a) Relative abundance of glutamate family amino acids, (b) aspartate family amino acids, (c) aromatic amino acids, and (d) Ser as well as Leu were displayed in column plots (SE, n = 4). Different letters above the bars indicate significant difference between treatments using one-way ANOVA test in SPSS software at P < 0.05. Concentrations (nmol g FW−1) of individual amino acids are reported in Supplemental Table S1.

    • We carried out clustering analysis and PCA with amino acid data to better dissect the effects of different N forms on the petal amino acid profile (Fig. 6). First, via clustering analysis, amino acids could be categorized into two groups. Group one included Asp, Asn, Ile, Leu and Arg whose content were the highest at the S1 stage with NH4NO3 treatment. Group two including Phe, Ser, Thr, Tyr, Trp, Glu, Gln, Pro and Lys, on the other hand, increased in their content at the S2 and S3 stages with NH4NO3 treatment. We then could clearly observe that both NH4-N and NO3-N treatment significantly influenced the petal amino acid profile relative to that with NH4NO3 treatment (Fig. 6a). PCA with amino acid data provided more information (Fig. 6b). Except S2, the petal amino acid profile in NH4-N was much closer to that in NH4NO3 at both the S1 and S3 stages. Taken together, different forms of N could greatly affect the amino acid profile in Petunia petals. However, relative to NO3-N treatment, total content of amino acids and changes in content of individual amino acids in NH4-N treated petals were closer to those with NH4NO3 treatment.

      Figure 6. 

      Further analysis of amino acid data. Heatmap representation of concentrations of 14 amino acids at (a) S1−S3 stages and (b), (c), (d) PCA of amino acids at each developmental stage. Colors (green to purple) and transparency of arrows toward each parameter indicate their contributions to the principal components.

    • Correlation analysis with petal color traits and amino acid profile data indicated that changes in the content of some amino acids were connected to petal color traits (Fig. 7). Thr, Tyr and Trp was positively correlated with the hue angle () of Petunia petals. Thr and Leu were negatively correlated with the yellowness/blueness (b*) and chroma (C*) whereas Tyr was negatively correlated with b*. These data suggested that when the petal amino acid profile was modified by different forms of N, the color traits such as color and brightness of petals could change accordingly.

      Figure 7. 

      Correlation analysis of L*, a*, b*, c*, and 14 amino acids. Data were processed via R programming. The intensity of blue and red color as well as the size of each dot are proportional to the calculated correlation coefficient (r).

      Considering we collected these data from a white-flower cultivar 'Mitchell Diploid', we purchased four commercially available cultivars of Petunia with four flower colors (Fig. 8a, b) and treated them in the same way to further analyze various forms of N on Petunia petals with colors. Petal color traits including L*, a*, b*, C* and of white-, pink-, dark pink- and red-flower Petunia plants treated with NH4NO3, NH4-N, and NO3-N were analyzed and presented in Supplemental Table S2. Interestingly, regardless of petal colors, relative to NH4NO3 treatment, both NH4-N and NO3-N treatment could influence L*. For pink-flower cultivar, NH4-N and NO3-N treatments slightly decreased a*, b* and C*. And for dark pink- and red-flower cultivars, NH4-N and NO3-N treatments slightly increased a*, b* and C* (Supplemental Fig. S1). PCA of petal color traits for each cultivar are shown in Fig. 8cf and revealed more information about the different treatments. For the white-flower cultivar, similar to data we collected from 'Mitchell Diploid', NH4-N and NO3-N could be clearly separated from NH4NO3 with a*, b* and C* contributing the most to separation (Fig. 8c). But NH4-N and NO3-N could not be separated from each other. For the pink-flower cultivar, NH4-N could be partially separated from NO3-N in PC2 with b* and contributed the most (Fig. 8d). And for the dark pink-flower cultivar, NH4-N could be completely separated from NO3-N in PC2 with a* and C* contributed the most (Fig. 8e). For the red-flower cultivar, NH4-N could be partially separated from NO3-N in PC2 with b* and contributed the most. To be noted, NO3-N could be completely separated from NH4NO3 in PC1 (Fig. 8f). Taken together, different forms of N could impact petal color traits. And for petals with colors, NH4-N and NO3-N could affect the redness/greenness and yellowness/blueness but the lightness of petals contributed less to differences between treatments.

      Figure 8. 

      Relations between amino acids and petal color traits of Petunia cultivars with different colors. (a), (b) Plant and petal appearance of four Petunia cultivars (Scale bar = 1 cm). (c)−(f) PCA of L*, a*, b*, C* and in each cultivar. Colors (green to purple) and transparency of arrows toward each parameter indicate their contributions to the principal components.

    • Ornamental traits such as size of flowers and color of petals are fundamentally determined by the metabolic status on the whole-plant level. Extensively performed agricultural practice and continuously increased concentration of CO2 in the atmosphere have both aggravated N conditions in soil. This study has been designed to specifically investigate the effects of ammonium or nitrate as the sole source of inorganic N on the developmental and chemical features of floral tissues in Petunia.

      According to the total N content on the whole-plant level after each treatment (Fig. 1a), we think that the Petunia x hybrida cv 'Mitchell Diploid' plants we used in this study do not have preferences to a specific form of inorganic N. To be noted, given the same amount of inorganic N (2 mM), plants treated with NO3-N alone accumulated more biomass. Previous studies[28] pointed out that responses of Petunia plants to different forms of inorganic N depended on the concentrations of N applied to plants and the medium used for plant growth. Ammonium is more effective than nitrate on the growth promotion in rice[29] and maize[30]. Complex effects of ammonium and nitrate on plant growth, CO2 assimilation and activities of key enzymes involved in N metabolism have also been reported in blueberry, raspberry and strawberry[31]. Similarly, different ratios of nitrate to ammonium applied to sweet pepper at different developmental stages bring various effects on flowering, fruit set and yield[32]. It is then clear to us that effects of different forms of inorganic N on ornamental plants should be discussed case by case[17].

      We found that in Petunia plants, NH4-N and NO3-N treatment could both enhance the number of branches and flowers at the cost of leaf and corolla area. Plants can generally benefit from the direct application of NH4-N at an appropriate level since the assimilation of ammonium is far more energetically efficient relative to nitrate assimilation. Meanwhile, nitrate is more mobile which can compensate this limitation. Therefore, applications of NH4-N and NO3-N alone could be superior to NH4NO3 treatment. N can shape the architecture of plants. Nitrate regulates shoot branching on the molecular level to control the outgrowth of axillary buds[33]. Nitrate can also control the transition from the vegetative to reproductive phase and therefore, is involved in flowering time regulation[34]. On the other hand, ammonium application usually leads to accumulation of proteins which is generally beneficial to crop growth. However, there are clear differences between the two treatments. Studies on tobacco have also shown that ammonium nitrogen treatment results in a reduction in leaf area[35]. 15N labeling experiments confirmed that only NH4-N changed the distribution of 15N between various tissues with floral tissues absorbing and utilizing more 15N. Meanwhile, relatively smaller amounts of 15N was distributed into the roots and leaves in NH4-N treated plants. Taken together, when ammonium is used as the sole source of inorganic N, Petunia plants can manage to import more N into floral organs and produce more flowers with smaller size. More importantly, both NH4-N and NO3-N treatment can modify the amino acid profile on the whole-plant level. And as the basic compounds for plant development and signaling molecules[36], amino acids may play direct roles in the formation of plant architecture via regulating the meristem tissues[37], which requires further study.

      Here we need to clarify that in most cases, ammonium (NH3 or NH+4) is a stress cue[38,39]. However, ammonium nutrition can also be advantageous as it can improve crop quality[40]. For example, when grown under ammonium nutrition, compared with nitrate, wheat grains can accumulate more proteins leading to an increased wheat bread-making quality[41]. This is somehow consistent with what we have found in Petunia plants. Petal color traits and amino acid profiles measured in this study are highly informative. For Petunia x hybrida 'Mitchell Diploid' with white flowers (Fig. 3), NH4-N and NO3-N treatments could significantly increase petal yellowness/blueness (b*) and chroma (C*) and decrease lightness (L*) and hue angle (). We got very similar data with the commercially available white-flower cultivar (Fig. 8c). And data collected for commercially available cultivars with pink-, dark pink- and red-flowers, further indicated that effects of NH4-N and NO3-N on petal pigmentation could be different when petal colors differed. Further studies should be carried out to determine an appropriate ratio of ammonium to nitrate which can be applied to ornamental plants to modify their petal colors.

      For amino acids in petals, there are few studies available. Roles of amino acids in flower development and senescence have been briefly reported in Eustoma grandiflorum[42]. Although most local amino acids in floral tissues are imported from roots and leaves, it has been recently proposed that some amino acids can be synthesized in flowers[18]. For example, de novo synthesis of Asn may exist in flowers due to the fact that asparagine synthetase1 (ASN1) is expressed abundantly in Arabidopsis flowers[43]. The biological roles of Asn in floral tissues can be important. On one hand, Asn carries more N than other amino acids and serves as a good N sink[44]. On the other hand, hydrolysis of Asn can release ammonium which in turn can be used to synthesize Gln[45,46]. More importantly, amino acids provide precursors and energy for petal color and scent. We think the metabolic roles of amino acids in regulating ornamental traits should be much more emphasized in this field. Accumulation of free amino acids is usually considered a sign for ammonium stress[47]. The fact that total content of 14 amino acids in NH4-N petals were not higher than those in NH4NO3 indicates that the NH4-N in our study was not a stress treatment. Given that the assimilation of inorganic N into amino acids primarily occur in roots, the 15N label data and petal amino acid profiles collected in this study indicated that Petunia plants treated with NH4-N alone loaded relatively more amino acids such as Glu, Gln, Asp and Asn to their flowers (Fig. 5a, b). We also found that changes in petal amino acid profiles were correlated with changes in their color traits (Fig. 7), which should be further investigated at the molecular level in ornamental plants.

    • In summary, we have analyzed effects of ammonium and nitrate alone on plant growth, the tissue-level N utilization and distribution, petal color traits and petal amino acid profiles in Petunia plants. We have confirmed that NH4-N can increase 15N distribution to floral tissues, modify the amino acid profiles in petals which in turn bring changes to petal colors. Mechanisms of governing flower development by different forms of N or other environmental cues require further investigation and will definitely lead to a better understanding of the metabolic regulation of ornamental traits.

      • We gratefully acknowledge the support of National Key Research and Development Program of China (2018YFD1000405 to F.M.). We thank College of Horticulture Science and Engineering at Shandong Agricultural University for supporting the Experimental Core Facilities where we performed mass spectrometry and phenotypic analysis. We thank State Key Laboratory of Crop Biology at Shandong Agricultural University for providing lab space and plant growth chambers. This work was supported by a grant from National Key Research and Development Program of China (2018YFD1000405) to Fangfang Ma.

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

      • Supplemental Table S1 Concentrations of 14 free amino acids in Petunia petals at three developmental stages measured via GC-MS for each treatment (SE, n=4). Different letters in each row indicate significant difference between treatments using one-way ANOVA test in SPSS software at P < 0.05.
      • Supplemental Table S2 CIELab coordinates including perceptual lightness (L*), redness/greenness (a*), yellowness/blueness (b*), chroma (C*), and hue angle (h°) in petals of four petunia cultivars measured for each treatment via a portable colorimeter. Detailed data of four biological replicates for each treatment were presented here.
      • Supplemental Fig. S1 Effects of ammonium-nitrate (NH4NO3), ammonium (NH4-N), and nitrate (NO3-N) on petal color traits in four commercially available petunia cultivars. Changes in CIELab coordinates including L*, a*, b*, C*, and h° in white- (a), pink- (b), dark pink- (c) and red-flower cultivar (d) were plotted in column figures (SE, n=4; scale bar = 1cm). Different letters above the bars in each column indicate significant difference between treatments using one-way ANOVA test in SPSS software at P < 0.05.
      • Copyright: © 2022 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 (8)  References (47)
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    Guo H, Wang G, Zhang P, Wang G, Bao Z, et al. 2022. Ammonium and nitrate impact petal color traits and amino acid profiles differently in Petunia. Ornamental Plant Research 2:14 doi: 10.48130/OPR-2022-0014
    Guo H, Wang G, Zhang P, Wang G, Bao Z, et al. 2022. Ammonium and nitrate impact petal color traits and amino acid profiles differently in Petunia. Ornamental Plant Research 2:14 doi: 10.48130/OPR-2022-0014

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