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2022 Volume 2
Article Contents
Abstract
Introduction
Structure and distribution of main bioactive constituents of lonicera
Biosynthesis and regulation of the main bioactive constituents of lonicera
Biosynthesis of chlorogenic acids and flavonoids
Biosynthesis of iridoids and triterpene saponins
Metabolic engineering of the main bioactive compounds of the lonicera genus
Metabolic engineering of chlorogenic acid, luteolin and their glycosides
Metabolic engineering of iridoids and triterpene saponins
Pharmacological activities of the main bioactive compounds of lonicera
Anti-inflammatory effects
Antimicrobial effects
Anti-oxidative stress effect
Hepatoprotection
Other pharmacological effects
Chloroplast genomic and phylogenetic relationships of lonicera species
Conclusions and future perspectives
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
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Contrasting responses to drought stress between Chrysanthemum japonense and C. nankingense

  • State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Flower Biology and Germplasm Innovation, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, 210095 Nanjing, China

  • # These authors contributed equally: Yi Zhang, Jing Gu

More Information
  • The response of Chrysanthemum japonense and C. nankingense to drought stress induced by polyethylene glycol was characterized at the level of leaf water status, leaf surface morphology and cuticular wax (quantity and composition), the activity of antioxidant enzymes, the extent of membrane lipid peroxidation, the accumulation of proline, photosynthesis performance and abscisic acid (ABA) accumulation. The more tolerant species C. japonense maintained its water status more effectively than C. nankingense, probably because its leaves form more cuticular wax and are able to accumulate higher levels of ABA. Superoxide dismutase activity was higher in C. japonense than in C. nankingense, as was that of catalase and ascorbate peroxidase during the later part of the stress episode, but levels of peroxidase were not differentiated at the end of the stress period. Membrane damage, as measured by electrolyte leakage and malondialdehyde accumulation, was less severe in C. japonense, which was also able to generate higher levels of free proline after a 10 h exposure to stress. Thus the superior response of C. japonense also reflects a more adapted system of osmoprotection and antioxidation. As a result, photosynthesis was compromised less by drought stress in C. japonense than in C. nankingense. That provides a scientific basis for the development and application of drought tolerance resources of chrysanthemum.

  • Introduction

    The Lonicera Linn. genus is a constituent member of the Caprifoliaceae family[1]. It is the largest genus in this family and comprises at least 200 species with a notable presence in North Africa, North America, Asia, and Europe[1]. Members of the Lonicera genus possess a wide range of economic benefits from their use as ornamental plants to food and as plants credited with numerous health benefits. Conspicuous among the numerous members of this genus with known medicinal uses are L. japonica, L. macranthoides, L. hypoglauca, L. confusa, and L. fulvotomentosa[2]. Though these species feature prominently in the Chinese Pharmacopoeia, other species such as L. acuminata, L. buchananii, and L. similis are recognized as medicinal resources in certain parts of China[1]. Among the aforementioned species, L. japonica takes precedence over the rest due to its high medicinal and nutritional value[3,4]. For instance, the microRNA MIR2911, an isolate from L. japonica, has been reported to inhibit the replication of viruses[5−7]. Also, the water extract of L. japonica has been used to produce various beverages and health products[8]. The Lonicera genus therefore possesses huge prospects in the pharmaceutical, food, and cosmetic industries as an invaluable raw material[9].

    The main active constituents of the Lonicera genus include organic acids, flavonoids, iridoids, and triterpene saponins. Chlorogenic acids, iridoids, and flavones are mainly credited with the anti-inflammatory, antiviral, anticancer, and antioxidant effects of the various Lonicera species[10−13]. Their hepatoprotective, immune modulatory, anti-tumor and anti-Alzheimer’s effects are for the most part ascribed to the triterpene saponins[14−16]. As stated in the Chinese Pharmacopoeia and backed by the findings of diverse research groups, the plants of the Lonicera genus are known to possess high amounts of organic acids (specifically chlorogenic acid) and pentacyclic triterpenoid saponins[2,17−19]. The flower and flower bud have traditionally served as the main medicinal parts of the Lonicera genus even though there is ample evidence that the leaves possess the same chemical composition[20]. A perusal of the current scientific literature reveals the fact that little attention has been devoted to exploring the biosynthesis of the chemical constituents of the Lonicera genus with the view to finding alternative means of obtaining higher yields. It is therefore imperative that priority is given to the exploration of the biosynthesis of these bioactive compounds as a possible means of resource protection. There is also the need for further research on ways to fully tap the medicinal benefits of other plant parts in the Lonicera genus.

    Here, we provide a comprehensive review of relevant scientific literature covering the structure, pharmacology, multi-omics analyses, phylogenetic analysis, biosynthesis, and metabolic engineering of the main bioactive constituents of the Lonicera genus. Finally, we proffer suggestions on the prospects of fully exploiting and utilizing plants of the Lonicera genus as useful medicinal plant resources.

    Structure and distribution of main bioactive constituents of Lonicera

    A total of at least 400 secondary metabolites have been reported for the Lonicera genus. These metabolites are categorized into four main groups (Fig. 1a), including not less than 50 organic acids, 80 flavonoids, 80 iridoids, and 80 triterpene saponins[21−23]. Organic acids are mainly derivatives of p-hydroxycinnamic acid and quinic acid. Among the organic acids, chlorogenic acids are reported to be the main bioactive compounds in L. japonica[24−26]. The organic acids are most abundant in the leaves, while the least amounts are found in the stem of L. japonica. The flowers of the plant are known to contain moderately high amounts of organic acids[27]. The basic core structure of the flavonoids is 2-phenylchromogen. Luteolin and its glycoside which are characteristic flavonoids of the Lonicera genus are most abundant in L. japonica[28]. On the whole, the flavonoid contents in L. japonica are also highest in the leaves, available in moderate amounts in the flowers, and in lowest amounts in the stem[21]. The core structures of the iridoids are iridoid alcohols, the chemical properties of which are similar to hemiacetal. The iridoids often exist in the form of iridoid glycosides in plants. Secoiridoids glycosides are predominant in the Lonicera genus[25]. In L. japonica, the contents of the iridoids are most abundant in the flowers, moderate in leaves, and lowest in the stem[21]. The characteristic saponins of the Lonicera genus are mainly pentacyclic triterpenoids, including the hederin-, oleanane-, lupane-, ursulane- and fernane-types, etc[22]. The hederin-type saponins are reported in the highest amounts in L. macranthoides[17] (Fig. 1b).

    Figure 1.  Core structures of main secondary metabolites and their distribution in five species of Lonicera. (a) 1 and 2, the main core structures of organic acids; 3, the main core structures of flavonoids; 4, the main core structures of iridoids; 5, the main core structures of triterpene saponins. (b) Comparison of dry weight of four kinds of substances in five species of Lonicera[17,28].
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    Biosynthesis and regulation of the main bioactive constituents of Lonicera

    Biosynthesis of chlorogenic acids and flavonoids

    The similarities between chlorogenic acid (CGA) and flavonoids can be traced back to their biosynthesis since p-coumaroyl CoA serves as the common precursor for these compounds[29]. p-coumaroyl CoA is obtained through sequential catalysis of phenylalanine and its biosynthetic intermediates by phenylalanine-ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate CoA ligase (4CL)[30−33].

    CGA is a phenolic acid composed of caffeic acid and quinic acid and is the most important bioactive compound among the organic acids. Its biosynthesis has been relatively well-established; three main biosynthetic routes have been propounded (Fig. 2a). One route relates to the catalysis of caffeoyl-CoA and quinic acid by hydroxycinnamoyl-CoA quinate transferase (HQT)/hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) to produce CGA[34−37]. The HQT-mediated pathway has been deemed the major route for CGA synthesis in in different plant species[38,39]. The second biosynthetic route stems from the biosynthesis of p-coumaroyl quinate through the catalytic effect of HCT/HQT and subsequent hydroxylation of p-coumaroyl quinate under the catalysis of p-coumarate 3'-hydroxylase (C3’H)[34,36,37]. For the third route, caffeoyl glucoside serves as the intermediate to form CGA, a process that is catalyzed by hydroxycinnamyl D-glucose: quinic acid hydroxycinnamyl transferase (HCGQT)[40,41].

    Figure 2.  Biosynthetic pathways of main bioactive constituents of Lonicera. (a) Biosynthetic pathways of chlorogenic acid. (b) Biosynthetic pathways of luteoloside. (c) Biosynthetic pathways of secologanin. (d) Biosynthetic pathways of hederin-type triterpene saponins. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl CoA ligase; HCT, hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase; C3’H, p-coumaroyl 3-hydroxylase; HQT, hydroxycinnamoyl-CoA quinate transferase; UGCT, UDP glucose: cinnamate glucosyl transferase; CGH, p-coumaroyl-D-glucose hydroxylase; HCGQT, hydroxycinnamoyl D-glucose: quinate hydroxycinnamoyl transferase; CHS, Chalcone synthase; CHI, Chalcone isomerase; FNS, Flavone synthase; F3H, flavonoid 30-monooxygenase/flavonoid 30-hydroxylase; UF7GT, flavone 7-O-β-glucosyltransferase; GPS, Geranyl pyrophosphatase; GES, geraniol synthase; G8O, geraniol 10-hydroxylase/8-oxidase; 8HO, 8-hydroxygeraniol oxidoreductase; IS, iridoid synthase; IO, iridoid oxidase; 7DLGT, 7-deoxyloganetic acid glucosyltransferase; 7DLH, 7-deoxyloganic acid hydroxylase; LAMT, loganic acid O-methyltransferase; SLS, secologanin synthase; FPS, farnesyl pyrophosphate synthase; SS, squalene synthase; SE, squalene epoxidase; β-AS, β-amyrin synthase; OAS, oleanolic acid synthetase.
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    The key enzymes in the biosynthesis of p-coumaroyl CoA, and invariably CGA, thus, PAL, C4H, and 4CL have been established in diverse studies such as enzyme gene overexpression/knockdown[42], enzyme activity analysis[33] and transcriptomics[18]. However, the centrality of HQT in the biosynthesis of CGA remains disputable. While some studies have reported a strong correlation between HQT expression level with CGA content and distribution[18,35,39,43,44], others found no such link[45], bringing into question the role of HQT as a key enzyme in CGA biosynthesis.

    Few studies have been conducted on the regulation of CGA biosynthesis in the Lonicera genus. It was found that overexpression of the transcription factor, LmMYB15 in Nicotiana benthamiana can promote CGA accumulation by directly activating 4CL or indirectly binding to MYB3 and MYB4 promoters[46]. LjbZIP8 can specifically bind to PAL2 and act as a transcriptional repressor to reduce PAL2 expression levels and CGA content[47]. Under NaCl stress, increased PAL expression promoted the accumulation of phenolic substances in leaves without oxidative damage, a condition that was conducive to the accumulation of the bioactive compounds in leaves[48].

    Luteolin and its derivative luteolin 7-O- glucoside (luteoloside) are representative flavonoids of the Lonicera genus. Similar to CGA, luteolin is biosynthesized from p-coumaroyl CoA but via a different route. The transition from p-coumaroyl CoA to luteolin is underpinned by sequential catalysis by chalcone synthetase (CHS), chalcone isomerase (CHI), flavonoid synthetase (FNS), and flavonoid 3'-monooxygenase/flavonoid 3'-hydroxylase (F3'H)[45,49,50] (Fig. 2b). Luteoloside is synthesized from luteolin by UDP glucose-flavonoid 7-O-β-glucosyltransferase (UF7GT)[51]. Similar to CGA biosynthesis, the key enzymes of luteolin synthesis include PAL, C4H, and 4CL in addition to FNS[33,45,52]. The content of luteoloside was found to be highly abundant in senescing leaves relative to other tissues such as stem, flowers, and even young leaves[52]. Through transcriptomic analysis, luteoloside biosynthesis-related differentially expressed unigenes (DEGs) such as PAL2, C4H2, flavone 7-O-β-glucosyltransferase (UFGT), 4CL, C4H, chalcone synthase 2 and flavonoid 3'-monooxygenase (F3'H) genes were found to be upregulated in the senescing leaves. Biosynthesis-related transcription factors such as MYB, bHLH, and WD40 were also differentially expressed during leaf senescence[52], while bHLH, ERF, MYB, bZIP, and NAC were differentially expressed during flower growth[53]. Further analysis of the transcription factors revealed that MYB12, MYB44, MYB75, MYB114, MYC12, bHLH113, and TTG1 are crucial in luteoloside biosynthesis[52,53].

    Biosynthesis of iridoids and triterpene saponins

    The biosynthesis of terpenoids mainly involves three stages; formation of intermediates, formation of basic structural skeleton, and modification of basic skeleton[54]. The intermediates of terpenoids are mainly formed through the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, and eventually converted to the universal isoprenoid precursors, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) through a series of enzyme-catalyzed reactions. Under the catalysis of geranyl pyrophosphatase (GPS), IPP is then converted to geranyl pyrophosphate (GPP). Different terpenoids are subsequently derived from GPP as the intermediate product. For instance, in the formation of secoiridoid, GPP first removes the phosphoric acid group to obtain geraniol, second through a series of reactions such as oxidation and cyclization, the skeleton of iridoid, namely iridodial, can be obtained. Finally, through a series of reactions, the basic carbon skeleton of the secoiridoid, namely secologanin, is obtained[55−61] (Fig. 2c). In the formation of triterpene saponins, the key step lies in the formation of the precursor, 2,3-oxidosqualene, a reaction that is catalyzed by squalene epoxidase (SE). There are many pentacyclic triterpenes in the Lonicera genus, the most important type being the hederin-type saponins with hederagenin as aglycones. Hederin-type saponins are produced after the synthesis of oleanolic acid from β-amyrin and catalyzed by β-starch synthetase (β-AS) and Oleanolic acid synthase (OAS)[62,63]. The skeletal modification of the triterpenoid saponins is mainly achieved via the activities of the CYP450 enzymes and UDP-glycosyltransferase (UGT). Hence, the corresponding aglycones are first obtained via oxidation by the CYP450 enzymes (e.g., CYP72A), and further subjected to glycosylation by the appropriate UGT enzyme[63−65] (Fig. 2d). Skeletal formations of the iridoids and triterpene saponins in general have been well researched, but the same cannot be said about the enzymes involved in biosynthesis of these groups of compounds in the Lonicera genus. To fully utilize the iridoids and triterpene saponins in the Lonicera genus, it is necessary to further explore their biosyntheses with the view to enhancing and optimizing the process.

    Metabolic engineering of the main bioactive compounds of the Lonicera genus

    Given the importance of the bioactive compounds in the Lonicera genus, continual isolation of these compounds using the traditional methods are not only tedious and time-consuming, but also unsustainable. With the development and application of microbial metabolic engineering, different strategies have been introduced to produce these bioactive compounds by heterologous synthesis (Table 1).

    Table 1.  Biosynthesis of Lonicera-specialized metabolites using metabolic engineering.
    Engineering bacteriaOperational methodsProductsYieldRefs
    S. cerevisiaeEliminate the tyrosine-induced feedback inhibition, delete genes involved in competing pathways and overexpress rate-limiting enzymesCaffeic acid569.0 mg/L[69]
    S. cerevisiaeEmploye a heterologous tyrosine ammonia lyase and a 4HPA3H complex composed of HpaB and HpaC derived from different speciesCaffeic acid289.4 mg/L[73]
    S. cerevisiaeSupply and recycle of three cofactors: FADH2, S-adenosyl-L-methion, NADPHCaffeic acid
    Ferulic acid    		Caffeic acid: 5.5 g/L;
    Ferulic acid: 3.8 g/L    		[117]
    E. coliKnocking out competing pathwaysCaffeic acid7,922 mg/L[118]
    E. coliArtificial microbial community, a polyculture of three recombinant Escherichia coli strainsChlorogenic acid250 μM[68]
    Cell-free biosynthesisExtract and purify spy-cyclized enzymes (CFBS-mixture)Chlorogenic acid711.26 mg/L[70]
    S. cerevisiaeThree metabolic engineering modules were systematically optimized: shikimate pathway and carbon distribution, branch pathways, CGA pathway genesChlorogenic acidFlask fermentation: 234.8 mg/L;
    Fed-batch fermentation:
    806.8 mg/L    		[119]
    E. coliUsing modular coculture engineering: construction of the defective strain improves the production and utilization of precursor substancesChlorogenic acid131.31 mg/L[122]
    E. coliIntroduce heterologous UDP-glucose biosynthetic genesLuteolin34 mg/L[120]
    Y. lipolyticaOverexpression of the key genes involved in the mevalonate pathway, the gene encoding cytochrome P450 (CYP716A12) to that encoding NADPH-P450 reductaseOleanolic acid129.9 mg/L[85]
    S. cerevisiaeImprove the pairing efficiency between Cytochrome P450 monooxygenase and reductase and the expression level of key genesOleanolic acid606.9 mg/L[121]
    S. cerevisiaeHeterologous expression and optimization of CrAS, CrAO, and AtCPR1, and regulation of ERG1 and NADPH regeneration systemOleanolic acid433.9 mg/L[123]
     | Show Table
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    Metabolic engineering of chlorogenic acid, luteolin and their glycosides

    Due to the demand for CGA in the food, pharmaceutical, chemical, and cosmetic industries, the traditional means of obtaining the same requires a relatively longer period for plant maturation to obtain low yields of the desired product. This therefore brings into question the sustainability and efficiency of this approach. The alternative and sustainable approach has been to produce CGA using synthetic biology and metabolic engineering.

    Current research has sought to utilize Escherichia coli (and its mutant strain) and Saccharomyces cerevisiae to synthetically generate CGA and other flavonoids[66−73]. For instance, Cha et al. employed two strains of E. coli to produce a relatively good yield of CGA (78 mg/L). Their approach was based on the ability of one strain to generate caffeic acid from glucose and the other strain to use the caffeic acid produced and quinic acid as starting materials to synthesize CGA[66]. Using a bioengineered mutant of E. coli (aroD mutant), Kim et al. increased the yield of CGA to as high as 450 mg/L[67]. Others have sought to increase the yield of CGA by employing a polyculture of three E. coli strains that act as specific modules for the de novo biosynthesis of caffeic acid, quinic acid and CGA. This strategy eliminates the competition posed by the precursor of CGA (i.e., caffeic acid and quinic acid) and generally results in improved production of CGA[68]. Saccharomyces cerevisiae is a chassis widely used for the production of natural substances from plants with an intimal structure that can be used for the expression of cytochrome P450 enzymes that cannot be expressed in E. coli. Researchers have used yeast to increase the production of organic acids[69]. A de novo biosynthetic pathway for the construction of CGA in yeast has been reported new cell-free biosynthetic system based on a mixture of chassis cell extracts and purified Spy cyclized enzymes were adopted by Niu et al. to a produce the highest yield of CGA reported so far up to 711.26 ± 15.63 mg/L[70].

    There are many studies on the metabolic engineering for the synthesis of flavonoids, but few on luteolin and its glycosides. Strains of E. coli have been engineered with specific uridine diphosphate (UDP)-dependent glycosyltransferase (UGT) to synthesize three novel flavonoid glycosides. These glycosides were quercetin 3-O-(N-acetyl) quinovosamine (158.3 mg/L), luteolin 7-O-(N-acetyl) glucosaminuronic acid (172.5 mg/L) and quercetin 3-O-(N-acetyl)-xylosamine (160.8 mg/L)[71]. Since most of the flavonoid glycosides synthesized in E. coli are glucosylated, Kim et al. in their bid to synthesize luteolin-7-O-glucuronide, deleted the araA gene that encodes UDP-4-deoxy-4-formamido-L-arabinose formyltransferase/UDP-glucuronic acid C-4'' decarboxylase in E.coli and were able to obtain a yield of 300 mg/L of the desired product[72].

    Metabolic engineering of iridoids and triterpene saponins

    Terpenoidal saponins are mostly derived from slow-growing plants and usually possess multiple chiral centers[74]. Traditional isolation and even chemical synthesis of the terpenoidal saponins are both tedious and uneconomical for large-scale production. Therefore, it is necessary to find other ways to synthesize these compounds known to have diverse pharmacological functions.

    Heterologous synthesis has become an important way to improve the target products. With the development of synthetic biology, heterologous synthesis of triterpene saponins involves chassis of both plant and microbial origin. In this regard, Nicotiana benthamiana is a model plant species for the reconstruction of the biosynthetic pathways of different bioactive compounds including monoterpenes, hemiterpenes, and diterpenes[59,75−77]. Aside from Nicotiana benthamiana, other plants have also been used as heterologous hosts[78]. Heterologous synthesis using microbial hosts mainly involves Saccharomyces cerevisiae and Escherichia coli[79−81], and other microorganisms[82,83]. Comparatively, plants as biosynthetic hosts have the advantages of an established photosynthetic system, abundant supply of relevant enzymes, and presence of cell compartments, etc. They are however not as fast growing as the microorganisms, and it is also difficult to extract and separate the desired synthesized compounds from them as hosts.

    Although heterologous synthesis has many advantages, the premise of successful construction of synthetic pathway in host is to elucidate the unique structure of the compound and the key enzyme reaction mechanism in the biosynthetic pathway. There is little research on metabolic engineering of the hederin-type pentacyclic triterpene saponins in Lonicera, but there are studies on the heterologous synthesis of its aglycone precursor, oleanolic acid[84,85]. There is a dearth of scientific literature on key enzymes in the biosynthesis of pentacyclic triterpenoid saponins in the Lonicera genus.

    Pharmacological activities of the main bioactive compounds of Lonicera

    Scientific evidence by diverse research groups has linked members of the Lonicera genus to a wide range of pharmacological effects (Fig. 3). These pharmacological effects are elicited by different chemical constituents, much of the underlying mechanisms of which have been elucidated by the omics techniques. Here, we summarize the pharmacological effects and pharmacodynamics of the Lonicera genus in the last 6 years.

    Figure 3.  Schematic summary of four main pharmacological effects (anti-inflammatory, antimicrobial, anti-oxidative and hepatoprotective effects) of the Lonicera genus and the underlying mechanisms of actions.
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    Anti-inflammatory effects

    Bioactive compounds of plants in the Lonicera genus have demonstrated varying degrees of anti-inflammatory actions. In a recent study, Lv et al. showed that lonicerin inhibits the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) through regulating EZH2/AtG5-mediated autophagy in bone marrow-derived macrophages of C57BL/6 mice[86]. The polysaccharide extract of L. japonica reduces atopic dermatitis in mice by promoting Nrf2 activation and NLRP3 degradation through p62[87]. Several products of Lonicera have been reported to have ameliorative effects on DSS-induced colitis. Among them, flavonoids of L. rupicola can improve the ulcerative colitis of C57BL/6 mice by inhibiting PI3K/AKT, and pomace of L. japonica can improve the ulcerative colitis of C57BL/6 mice by improving the intestinal barrier and intestinal flora[88,89]. The flavonoids can also ameliorate ulcerative colitis induced by local enema of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in Wistar rats by inhibiting NF-κB pathway[90]. Ethanol extract from L. Japonica has demonstrated the potential to inhibit the expressions of inflammatory cytokines in serum and macrophages of LPS-induced ICR mice[91]. The water extract of L. japonica and luteolin were found to exhibit their anti-inflammatory effects via the inhibition of the JAK/STAT1/3-dependent NF-κB pathway and induction of HO-1 expression in RAW263.7 cells induced by pseudorabies virus (PRV)[92].

    Antimicrobial effects

    Existing scientific evidence indicates that the extracts of plants in the Lonicera genus exhibit strong inhibition against different pathogenic microorganisms. Phenolic compounds from L. japonica demonstrated a particularly significant inhibitory effect against Staphylococcus aureus and Escherichia coli, in vitro, making these compounds potential food preservatives[93]. Influenza A virus is a serious threat to human health. Recent research has found the ethanol extract of L. japonica to possess a strong inhibitory effect against H1N1 influenza virus-infected MDCK cells and ICR mice[94]. The incidence of the COVID-19 pandemic called to action various scientists in a bid to find safe and efficacious treatment[95]. Traditional Chinese medicines became an attractive alternative in this search. The water extract of the flower bud of L. japonica which has traditionally served as a good antipyretic and antitussive agent attracted the attention of researchers. Scientific evidences have confirmed that the water extract of L. japonica can induce let-7a expression in human rhabdomyosarcoma cells or neuronal cells and blood of lactating mice, inhibiting the entry and replication of the virus in vitro and in vivo[96]. In addition, the water extract of L. japonica also inhibits the fusion of human lung cancer cells Calu-3 expressing ACE2 receptor and BGK-21 cells transfected with SARS-CoV-2 spike protein, and up-regulates the expression of miR-148b and miR-146a[97].

    Anti-oxidative stress effect

    Oxidative stress has been implicated in the pathophysiology of many diseases, hence, amelioration of the same could be a good therapeutic approach[98,99]. In keeping with this therapeutic strategy, various compounds from the Lonicera genus have demonstrated the ability to relieve oxidative stress due to their pronounced antioxidant effects. For instance, the polyphenolic extract of L. caerulea berry was found to activate the expression of AMPK-PGC1α-NRF1-TFAM proteins in the skeletal muscle mitochondria, improve the activity of SOD, CAT and GSH-Px enzymes in blood and skeletal muscle, relieve exercise fatigue in mice by reducing oxidative stress in skeletal muscle, and enhance mitochondrial biosynthesis and cell proliferation[100]. The diverse health benefits of the anthocyanins from L. japonica have been mainly credited to their antioxidant and anti-inflammatory effects. The anthocyanin and cyanidin-3-o-glucoside have been reported to possess the potential to prolong life and delay senescence of Drosophila through the activation of the KEAP1/NRF2 signaling pathway[101].

    Hepatoprotection

    The liver is an essential organ that contributes to food digestion and detoxification of the body. These functions expose the liver to diverse toxins and metabolites. The Lonicera genus is rich in phytochemicals that confer protection on the liver against various toxins. The phenolic compound, 4, 5-di-O-Caffeoylquinic acid methyl ester was shown to be able to improve H2O2-induced liver oxidative damage in HepG2 cells by targeting the Keap1/Nrf2 pathway[102]. Hepatic fibrosis is a complex dynamic process, with the propensity to progress to liver cancer in severe cases. The L. japonicae flos water extract solution increased the cell viability of FL83B cells treated with thioacetamide (TAA), decreased the levels of serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP), inhibited the transformation growth factor β1 (TGF-β1) and liver collagen deposition[103]. Sweroside, a secoiridoid glucoside isolate of L. japonica is known to protect the C57BL/6 mice liver from hepatic fibrosis by up-regulating miR-29a and inhibiting COL1 and TIMP1[104].

    Other pharmacological effects

    Aside from the aforementioned, other pharmacological effects have been ascribed to the Lonicera genus. The ethanolic extract of L. caerulea has been reported to inhibit the proliferation of SMMC-7721 and H22 hepatoma cells, while its anthocyanins induced the apoptosis of tumor cells via the release of cytochrome C and activation of caspase[105]. AMPK/PPARα axes play an important role in lipid metabolism. A chlorogenic acid-rich extract of L. Japonica was found to significantly decrease the early onset of high-fat diet-induced diabetes in Sprague-Dawley rats via the CTRPs-AdipoRs-AMPK/PPARα axes[106]. In a high-fat diet-induced non-alcoholic fatty liver disease in C57BL/6 mice, treatment with L. caerulea polyphenol extract decreased serum inflammatory factors and endotoxin levels and the Firmicutes/Bacteroidetes ratio, an indication of its modulatory effect on the gut microbiota[107]. The iridoid-anthocyanin extract from L. caerulea berry contributed to alleviating the symptoms of intestinal infection with spirochaeta in mice[108].

    Chloroplast genomic and phylogenetic relationships of Lonicera species

    The traditional classification of the Lonicera genus based on the morphology of member plants is further categorized into two subgenera, Chamaecerasus and Periclymenum. The Chamaecerasus includes four categories, Coeloxylosteum, Isika, Isoxylosteum and Nintooa. The Periclymenum includes two categories, Subsect. Lonicera and Subsect. Phenianthi (Supplemental Table S1).

    High-throughput chloroplast genome sequencing of L. japonica found its length to be 155078 bp, which is similar to the structure of the typical angiosperm chloroplast genome. It contains a pair of inverted repeat regions (IRa and IRb, 23774 bp), a large single copy region (LSC, 88858 bp) and a small single copy area (SSC, 18672 bp)[109,110]. However, compared with chloroplast genomes of other plants, the chloroplast genome of L. japonica has a unique rearrangement between trnI-CAU and trnN-GUU[110]. Based on the phylogenetic analysis of the plastid genomes of seven plants in the Lonicera genus, 16 diverging hot spots were identified as potential molecular markers for the development of the Lonicera plants[111]. The phylogeny of Lonicera is rarely researched at the molecular level and the pattern of repetitive variation and adaptive evolution of the genome sequence is still unknown. Chloroplast genome sequences are highly conserved, but insertions and deletions, inversions, substitutions, genome rearrangements, and translocations also occur and have become powerful tools for studying plant phylogeny[112,113].

    We present here the phylogenetic tree of the Lonicera genus based on the published complete chloroplast genome sequences downloaded from the National Center for Biotechnology Information (NCBI) database using the Maximum likelihood method (Fig. 4). Based on our chloroplast phylogenies, we propose to merge L. harae into Sect. Isika and L. insularis into Chamaecerasus, but whether L. insularis belongs to Sect. Isika or Sect. Coeloxylosteum is uncertain. Based on protein-coding regions (CDS) of the chloroplast genome or complete chloroplast genomes, Liu et al. and Chen et al. supported the classification of the two subgenera in Lonicera[111,114]. Sun et al. and Srivastav et al. demonstrated a classification between the two subgenera with more species by using sequences of nuclear loci generated, chloroplast genome, and restriction site-associated DNA sequencing (RADSeq)[115,116]. However, our phylogenetic analysis and that of Sun et al. show relations within the subgenus Chamaecerasus are tanglesome in some respects[116]. Plant traits are affected by the environment to varying degrees. Since evidence of plant speciation is implicit in its genome sequence, comparative analysis at the molecular level provides a relatively accurate depiction of inherent changes that might have occurred over time. These findings suggest the need for more species of the Lonicera genus to be sequenced to provide a more accurate theoretical basis for the evolution of the Lonicera plants and a more effective revision in the classification of the Lonicera genus.

    Figure 4.  Phylogenetic tree of 42 species of the Lonicera genus based on complete chloroplast genome sequence data. The phylogenetic tree was constructed by the maximum likelihood method. Coeloxylosteum, Isika, Isoxylosteum, and Nintooa belong to Chamaecerasus and Subsect. Lonicera belongs to the Periclymenum. Chamaecerasus and Periclymenum are the two subgenera of Lonicera. 'Not retrieved' indicates that the species failed to retrieve a subordinate taxon in the Lonicera.
    DownLoad: Full-Size Img PowerPoint

    Conclusions and future perspectives

    The Lonicera genus is rich in diverse bioactive compounds with immeasurable prospects in many fields. Members of this genus have been used for thousands of years in traditional Chinese medicine for heat-clearing and detoxification. These plants generally have a good taste and form part of the ingredients of various fruit juices. In cosmetics, they are known to possess anti-aging and moisturizing functions. Plants of the Lonicera genus are also known for their good ecological adaptability and can be used to improve soil and ecological environment. Based on the value of the Lonicera genus, besides researching their use through molecular biological means, their efficient utilization can also be promoted in the following ways: (1) The stems and leaves of the plants could be developed for consumption and use since the chemical profiles of these parts do not differ significantly from the flowers. This way, the wastage of this scarce resource could be minimized or avoided. (2) Most of the Lonicera plants are vines or shrubs and their natural regeneration speed is slow, so the introduction and domestication of species could be strengthened to avoid overexploitation of wild resources.

    At present, only the research on the biosynthesis and efficacy of chlorogenic acid is quite comprehensive and has been used widely in various fields. There is limited research on various aspects of other bioactive compounds and should therefore be given priority in future research goals. Currently, the multi-omics analytical approach has gradually evolved as a reliable and helpful analytical platform. Hence, multi-omics research on the Lonicera genus could lead to discoveries in drug discovery and human health.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and design, draft manuscript preparation: Yin X, Chen X, Li W, Tran LSP, Lu X; manuscript revision: Yin X, Chen X, Li W, Tran LSP, Lu X, Chen X, Yin X, Alolga RN; data/literature collection: Chen X, Yin X; figure preparation: Chen X, Yin X; figure revision: Alolga RN, Yin X, Chen X, Li W, Tran LSP, Lu X. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

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

    Acknowledgments

    This work was partially supported by the National Natural Science Foundation of China (NSFC, Nos 82173918 and 82373983).

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

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  • Cite this article

    Zhang Y, Gu J, Xia X, Zeng J, Sun H, et al. 2022. Contrasting responses to drought stress between Chrysanthemum japonense and C. nankingense. Ornamental Plant Research 2:16 doi: 10.48130/OPR-2022-0016
    Zhang Y, Gu J, Xia X, Zeng J, Sun H, et al. 2022. Contrasting responses to drought stress between Chrysanthemum japonense and C. nankingense. Ornamental Plant Research 2:16 doi: 10.48130/OPR-2022-0016
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Contrasting responses to drought stress between Chrysanthemum japonense and C. nankingense

  • State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Flower Biology and Germplasm Innovation, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, 210095 Nanjing, China

  • Received: 15 August 2022
  • Accepted: 21 September 2022
  • Published online: 25 October 2022
Ornamental Plant Research  2,  Article number: 16  (2022)  |  Cite this article

Abstract: The response of Chrysanthemum japonense and C. nankingense to drought stress induced by polyethylene glycol was characterized at the level of leaf water status, leaf surface morphology and cuticular wax (quantity and composition), the activity of antioxidant enzymes, the extent of membrane lipid peroxidation, the accumulation of proline, photosynthesis performance and abscisic acid (ABA) accumulation. The more tolerant species C. japonense maintained its water status more effectively than C. nankingense, probably because its leaves form more cuticular wax and are able to accumulate higher levels of ABA. Superoxide dismutase activity was higher in C. japonense than in C. nankingense, as was that of catalase and ascorbate peroxidase during the later part of the stress episode, but levels of peroxidase were not differentiated at the end of the stress period. Membrane damage, as measured by electrolyte leakage and malondialdehyde accumulation, was less severe in C. japonense, which was also able to generate higher levels of free proline after a 10 h exposure to stress. Thus the superior response of C. japonense also reflects a more adapted system of osmoprotection and antioxidation. As a result, photosynthesis was compromised less by drought stress in C. japonense than in C. nankingense. That provides a scientific basis for the development and application of drought tolerance resources of chrysanthemum.

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    INTRODUCTION

    • Plant growth is seriously affected by abiotic stresses such as drought, low temperature and soil salinity. Drought is of particular concern in view of the predicted consequences of global climate change[1]. Severe episodes of drought stress lead to a shut down of photosynthesis, disturb the plant's core metabolism and can lead to plant death[2]. Plants have evolved a range of strategies, such as physical (leaf surface morphology), biochemical adaption and transcriptional reprograming, to combat drought stress[3,4].

      In the process of long-term evolution, plants have formed a series of physical defenses with their own organizational structures to resist the damage of the external environment, such as trichome and waxy cuticles[5]. Trichomes are hairy appendages on the surface of plants, which protect plant tissues from insects and ultraviolet (UV), and increase the tolerance of plants to drought stress[6]. The development of the cuticle, comprising a lipid layer (cutin) intermeshed and coated with wax, is one of the major adaptations for withstanding short term drought stress[7]. The cutin molecule is composed of cross-linked C16 and C18 ω-hydroxyl fatty acids, while the wax is a complex mixture of long-chain fatty acids and their derived alcohols, aldehydes, alkanes, ketones, and wax esters[8,9]. An increased deposition of cuticular wax has been associated with higher levels of drought tolerance in both rice and Arabidopsis thaliana[10,11]. Under drought conditions, the phytohormone abscisic acid (ABA), a key regulator of leaf stomatal conductance, is triggered[12,13]. Due to increase of ABA level under drought, the guard cell forcibly closes the stomata to reduce transpirational water loss, and inhibits photosynthesis by preventing the entry of CO2[2,14,15]. At the same time, the shrinkage in cell volume caused by water shortage increases the viscosity of the cellular content, hindering normal enzymatic function as a consequence[16]. A drought stress-induced loss in photosynthetic activity can also generate oxidative stress on account of the build-up of reactive oxygen species (ROS)[17,18]. Under normal conditions, plants scavenge ROS by a range of enzymatic and non-enzymatic means[19]. The capacity to neutralize ROS has been associated with the level of drought tolerance in a number of plant species[20−22]. Some plant species also show a pronounced capacity to adjust the cellular osmotic environment in response to drought stress by accumulating highly soluble non-toxic compounds such as sugars (sucrose, trehalose and sorbitol), free amino acids (proline) and amines (glycine betaine and polyamines)[23,24].

      The ornamental species chrysanthemum (Chrysanthemum morifolium) is widely appreciated as a source of cut flowers and pot plants. Most chrysanthemum cultivars are very vulnerable to drought stress, but some of the wild relatives of C. morifolium have been identified as important reservoirs of genetic variation relevant for drought tolerance improvement[25,26]. Since the physiological response of Chrysanthemum spp. to drought stress is poorly understood, we set out to study leaf surface morphology and the response of key antioxidant enzymes, photosynthesis and endogenous levels of ABA to artificially induced drought stress in C. nankingense and C. japonense, two species characterized by a differential level of drought tolerance.

    MATERIALS AND METHODS

      Plant material and stress treatments

    • The accessions of C. nankingense (drought-sensitive) and C. japonense (drought-tolerant) were obtained from the Chrysanthemum Germplasm Resource Preserving Centre, Nanjing Agricultural University, China. Rooted cuttings (six leaf stage) were grown hydroponically in Hoagland solution (pH 5.8) under a 12 h photoperiod (300 µmol·m−2·s−1 photosynthetically active radiation), a relative humidity of 70% and a day/night temperature of 25/20 °C. The material was acclimated to these conditions for six days before the imposition of polyethylene glycol (PEG)-induced drought stress. Following the method of Zhang et al.[27], the plants were transferred for two, four, six, eight or 10 h into a solution of 20% w/v PEG 6000 dissolved in Hoagland, generating a potential of ~ −0.52 MPa. Control plants were retained in half strength Hoagland's solution (−0.01 MPa). The experiment was set out as a completely randomized split-plot with three replications (six plants per species per replication). The physiological and biochemical assays were conducted on the third or fourth leaves below the apex of the shoot.

      Water status

    • Leaf wilting was rated visually on a scale of zero (no observable wilting) to five (severely wilted)[27]. The relative water content (RWC) of leaves was estimated following the methods of Galmés et al.[28]. Each data point represented the mean of three independent leaves.

      Characterization of the leaf surface and its cuticular wax

    • The morphology of the leaf surface was observed by scanning electron microscopy, according to the methods of He et al.[29]. To calculate the density of trichome and stomata, each sample was observed under six different scope visual fields. Cuticular waxes were extracted from 0.2 g fully expanded leaves by incubating in 10 ml chloroform for 30 s at room temperature. An internal standard was provided by adding 5 µg n-tetracosane (C24) to each sample. The solvent was evaporated under a mild nitrogen stream, then redissolved in a mixture of 100 μl pyridine, 100 μl bis-N,N-(trimethylsilyl)-trifluoroacetamide (Macherey-Nagal, Düren, Germany). After heating at 70 °C for 1 h, the solvent was evaporated again under nitrogen and the samples redissolved in 200 μl chloroform. Qualitative and quantitative composition analyses followed the methods of Lee et al.[30]. A 1 μl aliquot was separated by GC–MS (Agilent 7890A-5975C, USA) and quantification was based on the internal standard.

      Enzyme assays

    • Leaf samples were stored at −80 °C after quick freezing in liquid nitrogen. The frozen leaf segments (0.25 g) were ground to a powder in liquid nitrogen, and soluble protein was extracted by homogenization in 1 ml 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% w/v polyvinyl pyrrolidone 40. The supernatant of centrifuged homogenate (12,000 g, 15 min, 4 °C) is directly used for subsequent enzyme analysis. Total protein content was determined according to the Bradford dye-binding method[31]. Superoxide dismutase (SOD) activity assay was performed following the method of Giannopolitis & Ries[32] with minor modifications. Each 3 ml reaction mixture (50 mM potassium phosphate buffer (pH 7.8), 13 mM L-methionine, 75 μM nitroblue tetrazolium (NBT), 2 μM riboflavin, 1 mM EDTA and 100 μl supernatant) was illuminated for 10 min in white fluorescent light (100 µmol·m−2·s−1). Then the SOD activity was measured at 560 nm. Peroxidase (POD) activity was measured by monitoring the increase in absorbance at 470 nm caused by the oxidation of guaiacol, which was slightly modified according to the method of Li[33]. Each 3 ml reaction was initiated by adding 20 μl 40 mM H2O2 into 2.9 ml 50 mM phosphate buffered saline (PBS) (pH 7.0), 50 μl 20 mM guaiacol and 30 μl supernatant. PBS was used as blank control instead of supernatant. The catalase (CAT) assay was based on method of Beers & Sizer[34] with minor modifications. Each 3 ml reaction was initiated by adding 50 mM potassium phosphate buffer (pH 7.0), 15 mM H2O2 and 100 μl supernatant. Ascorbate peroxidase (APX) activity was assayed following the method of Nakano & Asada[35] with minor modifications. Each 3 ml reaction was initiated by adding 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM H2O2 and 100 μl supernatant.

      Cell membrane stability, malondialdehyde (MDA) and free profine content

    • Cell membrane stability was determined by measuring electrolyte leakage (EL). Following the method of Hu et al.[36], whole fully expanded leaves were sliced and incubated in 10 ml distilled deionized water on a shaker for 24 h. The conductance of the solution at 24 h was taken as the initial level (Ci). Thereafter, the material heated to 100 °C for 10 min, and the conductance of the solution (Cmax) was determined again. The EL was calculated by the expression (Ci/Cmax) × 100%. For lipid peroxidation analysis, the MDA content was measured using the thiobarbituric acid (TBA) method described by Heath & Packer[37] with minor modifications. Fresh leaf tissue (0.5 g) was ground and extracted in 5 ml 5% w/v trichloroacetic acid (TCA). The homogenate was centrifuged (12,000 g, 5 min), and 2 ml of the supernatant was added to 2 ml 0.67% w/v TBA (prepared in 10% v/v TCA). The mixture was rapidly cooled after heating to 100°C for 30 min, and centrifuged (12,000 g, 10 min). The absorbance of the supernatant was monitored at 532 nm. Correction of non-specific turbidity was obtained by subtracting the absorbance value taken at 600 nm. The level of lipid peroxidation was expressed as nmol per g fresh weight. Free proline was extracted and determined as described by Bates et al.[38] with minor modifications.

      Chlorophyll content and photosynthesis related parameters

    • Chlorophyll (0.1 g) was extracted in 95% ethanol for 48 h and the absorbance of the supernatant detected at 470, 649 and 665 nm. The quantity of total chlorophyll (a + b) was determined as described by Li[33]. The net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci) of fully expanded leaves were monitored using a LI-COR 6400 portable photosynthesis system (LI-COR, Lincoln, NE, USA). The CO2 concentration in the chamber was 380 ± 10 µmol/mol and a photosynthetic photon flux density of 1000 µmol·m−2·s−1 at the leaf surface was provided by an LED red-blue light source (LI-COR 6400-02). The maximum quantum efficiency of PSII photochemistry (Fv/Fm) was determined in the same intact leaves according to the method of Liu et al.[39]. For each treatment, Pn, Gs, Tr, Ci and Fv/Fm values were obtained from five leaves at each time point.

      Endogenous ABA level

    • Frozen leaf (~1 g fresh weight) was ground in liquid nitrogen and homogenized for 12 h in 10 ml pre-cooled 80% v/v aqueous methanol under low light. The mixture was centrifuged (12,000 g, 4 °C, 10 min) and the pellet extracted twice in 10 ml 80% methanol at 4 °C under low light. The supernatant was filtered through a Sep-Pak C18 gel cartridge and freeze dried. The lyophilisate was redissolved in 1 ml methanol and passed through a 0.45 μm filter. Quantification of ABA was conducted by high performance liquid chromatography (HPLC) (Agilent Technologies 1100) as described by Ciha et al.[40] with minor modifications. The separation column was supplied by Agilent (HC-C18, 5 μm, 250 mm × 4.6 mm). The solvents were 0.6% v/v glacial acetic acid (A) and 100% methanol (B); the initial solvent was 100% A, moving to 50% A, 50% B over the subsequent 10 min, where it was held for 20 min. The solvent flow rate was 1 ml/min, the detection wavelength 254 nm and the column temperature 30 ± 0.2 °C. Quantification was based on calibration with known ABA standards (Sigma-Aldrich Chemie, Munich, Germany).

      Statistical analysis

    • All data are mean ± standard deviation (SD). IBM SPSS Statistics 17.0 software and Microsoft Excel 2007 was used for statistical analysis. A one-way analysis of variance, followed by Duncan’s multiple range test (with P set at 0.05/0.01), was employed to assess whether treatment means differed significantly from one another.

    RESULTS

      Plant drought stress phenotype and leaf water status

    • The wilting index of unstressed plants was zero, and the stress induced wilting in both species (Fig. 1a). After 2 h of stress, the wilting index of C. japonense rose to one, and the lower leaves had wilted and had begun to droop (Y1 in Fig. 1a). Wilting set in earlier and was more severe in C. nankingense. By 2 h, its wilting index had already reached two, and its lower leaves were wilted and drooping (N1 in Fig. 1a); after 10 h, the wilting index was five and all the leaves appeared dehydrated and withered (N5 in Fig. 1a). At this stage, the wilting index of C. japonense was still only three and its uppermost leaves remained turgid (Y5 in Fig. 1a).

      Figure 1. 

      The response of C. japonense and C. nankingense to PEG-induced drought stress. (a) The morphological response of C. japonense and C. nankingense to PEG-induced drought stress. Y0-Y5, N0-N5: C. japonense (Y) and C. nankingense (N) plants subjected to, respectively, 0, 2, 4, 6, 8 and 10 h of stress. The wilting index ranges from 0 (no observable wilting) to 5 (severely wilted). Scale bars = 1 cm. (b) The response of leaf RWC to PEG-induced drought stress. Y: C. japonense, N: C. nankingense, C: Control (no PEG), T: PEG treatment. ** Value significant at P ≤ 0.01. Values given as mean ± SD (n = 3).

      The RWC of both species was maintained at the same level under non-stressed conditions (Fig. 1b), but declined markedly as a result of the stress treatment. The decline was more acute in C. nankingense than in C. japonense. The RWC in the leaf of the latter was significantly higher than in the former after only 4 h of PEG treatment, while after a 10 h exposure, the RWCs had fallen to, respectively, 62.3% and 73.3%.

      Leaf surface morphology

    • A marked difference in the appearance of the leaf surface was observed between the two species. The trichome density on the upper and lower leaf surface of the C. nankingense leaf was low (0.10 and 1.79 per mm2 respectively) (Table 1), while in contrast, C. japonense developed many trichomes especially on the lower leaf surface - the density on the upper leaf surface was 33.45 per mm2, while that on the lower surface was too high to count. The abundance of trichomes prevented the measurement of stomatal density, but on the upper leaf surface, stomatal density in the C. japonense was significantly greater than on the equivalent C. nankingense leaf surface (76.57 vs 11.96 per mm2, respectively) (Table 1), and the C. japonense gland cells were larger than those on the C. nankingense leaf (Fig. 2d, h).

      Table 1.  Variation in leaf surface morphology in C. japonense and C. nankingense.

      SpeciesUpper epidermis of leafLower epidermis of leaf
      Trichome density (mm−2)Stoma density (mm−2)Trichome density (mm−2)Stoma density (mm−2)
      C. japonense33.45 ± 1.46A76.57 ± 11.72 A∞N
      C. nankingense0.11 ± 0.12B11.96 ± 10.81B1.79 ± 0.47346.94 ± 24.73
      Values (given as mean ± SD) labeled with a different letters differed significantly (P ≤ 0.01) (n = 6). ∞ means too much to calculate. N means unable to observe because of the well-developed trichome layer covering lower epidermis of leaf.

      Figure 2. 

      Scanning electron microscopic images of the leaf surface of C. japonense (a-d) and C. nankingense (e-h). (a) and (e): upper leaf surface, (b) and (f): lower leaf surface, (c) and (g): a single trichome, (d) and (h): a single stomate.

      Cuticular wax amount and composition

    • The total wax load on the C. japonense leaf was ~6.6 fold greater than on the C. nankingense leaf (Fig. 3a). There was also a significant difference between the species for cuticular wax composition. Fatty alcohols (include primary alcohols and secondary alcohols) were the predominant component (39.9%) of the C. japonense leaf wax, followed by esters (33.1%), alkanes (21.3%) and fatty acids (5.7%). In C. nankingense, fatty alcohols were even more predominant (49.8%), while the remainder was composed of alkanes (35.4%) and esters (14.8%). The level of fatty acids in C. nankingense cuticular wax was below the level of detection. Nine components were specific to the cuticular wax of C. japonense, namely C20 and C24 fatty acids, C14, C22 and C24 primary alcohols, and C16, C17, C31 and C32 esters. A C20 ester was the only component specific for C. nankingense. Eight components were shared: C26 and C28 primary alcohols, C30 secondary alcohol, C17, C24 and C32 alkanes, C30 ester (although its content was greater in C. japonense) and C30 primary alcohol (the content of this component was greater in C. nankingense) (Fig. 3b).

      Figure 3. 

      (a) Quantity and (b) composition of cuticular wax on the C. japonense (Y) and C. nankingense (N) leaf. ** Value significant at P ≤ 0.01. Bars indicate the SD of the mean (n = 3).

      Antioxidant enzyme activity

    • The PEG treatment enhanced the activity of SOD, POD, CAT, and APX in both species. SOD activity was greater in C. japonense than in C. nankingense throughout the stress treatment (Fig. 4a). In C. japonense, it rose to 2.0 fold its background level after 8 h exposure and to 1.6 fold after 10 h, while in C. nankingense, the equivalent levels were 1.1 and 1.2 fold. POD activity tended to be greater in C. nankingense, although after 4 h of treatment it reached 1.9 fold of the background level in C. japonense, representing 1.3 fold the C. nankingense level (Fig. 4b). The background level of CAT activity was higher in C. nankingense than in C. japonense. In response to PEG treatment, it increased markedly in both species (Fig. 4c), reaching 1.3 and 1.6 fold of the background level in C. nankingense after, respectively, 2 h and 4 h of treatment. In C. japonense, CAT activity rose to 1.2 and 1.8 fold of the background after 2 h and 4 h of treatment, respectively. After 6 h of exposure, activity had risen to 1.4 (C. nankingense) and 2.0 (C. japonense) fold of the background level, although these levels were not statistically different from one another. As the stress was prolonged, CAT activity in C. japonense rose to nearly two fold the background level, but in C. nankingense, the increase was much more modest. APX activity was also greater in C. nankingense than in C. japonense under non-stressed conditions (Fig. 4d). The PEG treatment rapidly induced APX activity in C. nankingense,while that in C. japonense increased slowly. APX activity in C. nankingense reached 1.7 fold of background by 8 h, and 1.4 fold by 10 h, while in C. japonense, the equivalent levels were 3.0 fold and 3.3 fold.

      Figure 4. 

      Enzymatic activity (SOD (a), POD (b), CAT (c), and APX (d)) in the leaf of droughted C. japonense (Y) and C. nankingense (N) plants. C: Control (no PEG), T: PEG treatment. *, ** Value significant at P ≤ 0.05 or 0.01. Values given as mean ± SD (n = 3). SD’s indicated by a bar.

      EL and MDA content

    • Under control conditions, EL was maintained at a constant low level in both species (Fig. 5a). However, when subjected to PEG treatment, it increased as the time of exposure was lengthened. The C. nankingense EL was significantly higher than that of C. japonense throughout the whole period. By the end of the stress period treatment, it had reached 3.4 fold the control level in C. japonense and 3.8 fold in C. nankingense. The leaf MDA content behaved in a similar fashion (Fig. 5b), increasing in both species as the plants were exposed to stress. The increase set in earlier and was more pronounced in C. nankingense. After 2 h, the MDA content in the C. japonense leaf was no different from the background level, while in C. nankingense it had risen by 1.3 fold. By the end of the stress treatment, the MDA content of the C. japonense and C. nankingense leaves were, respectively 1.7 and 2.7 fold that of the non-stressed controls, indicating that the membrane lipid of C. nankingense was highly peroxidized and the cell membranes system seriously damaged.

      Figure 5. 

      (a) Electrolyte leakage and (b) MDA content in droughted leaves of C. japonense (Y) and C. nankingense (N). C: Control (no PEG), T: PEG treatment. ** Value significant at P ≤ 0.01. Values given as mean ± SD (n = 3).

      Free proline content

    • The accumulation of proline was negligible under control conditions, but the PEG treatment induced a significant accumulation in proline. C. japonense responded to water deficient stress more quickly, and accumulated more proline than C. nankingense (Fig. 6). The proline content in the C. japonense leaf was 1.6 and 2.4 fold of background at 2 h and 4 h respectively, and the corresponding levels were 1.2 and 1.6 fold in C. nankingense. The proline level in the C. japonense leaf was higher than that in the C. nankingense leaf throughout the stress treatment.

      Figure 6. 

      Free proline content in droughted leaves of C. japonense (Y) and C. nankingense (N). C: Control (no PEG), T: PEG treatment. ** Value significant at P ≤ 0.01. Values given as mean ± SD (n = 3). SD's indicated by a bar.

      The effect of drought stress on photosynthetic parameters

    • Pn, Gs, Tr, Fv/Fm and chlorophyll content were negatively affected by drought stress in both species, while the Ci parameter increased. The background level of Pn in C. nankingense was ~8.7 μmol CO2 m−2·s−1, somewhat higher than in C. japonense. In plants subjected to stress, this parameter decreased more sharply in C. nankingense than in C. japonense (Fig. 7a). By 2 h, it had fallen to 0.7 (C. nankingense) and 0.9 (C. japonense) fold of the control, and remained higher in C. japonense than in C. nankingense during the rest of the treatment. By 10 h, it had fallen to 0.1 fold in C. japonense and close to zero in C. nankingense. Gs behaved in a similar way. It decreased more rapidly in C. nankingense than in C. japonense (Fig. 7b), and over the period 6−10 h, remained higher in C. japonense than in C. nankingense. Tr followed the same pattern. Under control conditions, it was higher in C. nankingense than in C. japonense (Fig. 7c), after 2 h of stress it had fallen to 0.8 fold the background in both species. As the stress was prolonged, Tr fell in C. nankingense to 0.6 (4 h), 0.3 (6 h) and 0.1 (8 h) fold of the background level, and in C. japonense to, respectively, 0.6, 0.4 and 0.3 fold at these time points. Under control conditions, the Ci of C. japonense was higher than that of C. nankingense. It increased significantly in C. nankingense in response to PEG treatment (Fig. 7d). In contrast, in C. japonense, it fell very slightly over the first four hours of stress, only rising above the background level thereafter. Its level was higher in C. nankingense than in C. japonense throughout the stress treatment. Under control conditions, the Fv/Fm ratio remained stable at > 0.8 (Fig. 7e); exposure to PEG stress had a negative effect on both species, particularly on C. nankingense. By the end of the treatment, the Fv/Fm of C. nankingense and C. japonense were, respectively 0.5 and 0.7 fold that of the background. Under control conditions, the chlorophyll content of the leaves of C. nankingense was significantly higher than in those of C. japonense, but it decreased more quickly in C. nankingense than in C. japonense when the plants were exposed to PEG treatment (Fig. 7f). By the end of the stress treatment, the chlorophyll content of C. nankingense was 0.6 fold and that of C. japonense was 0.8 fold the initial levels, and the chlorophyll content of C. japonense was significantly higher than that of C. nankingense.

      Figure 7. 

      Photosynthetic parameters (Pn (a), Gs (b), Tr (c), Ci (d), Fv/Fm (e) and chlorophyll (a + b) content (f)) in the droughted leaves of C. japonense (Y) and C. nankingense (N). C: Control (no PEG), T: PEG treatment. *, ** Value significant at P ≤ 0.05 or 0.01. Values given as mean ± SD (n = 5). SD’s indicated by a bar.

      Leaf ABA content

    • The ABA content of the leaves of both species was consistently low under control conditions (Fig. 8), but increased markedly in response to PEG treatment. The response of C. japonense plants was much larger than that of C. nankingense plants. The ABA content in the C. japonense leaves increased rapidly over the first six hours of stress, and thereafter more slowly. The ABA content in the leaves of C. japonense was 1.7, 2.3 and 1.5 fold higher than in the leaves of C. nankingense at 2 h, 6 h and 10 h respectively.

      Figure 8. 

      ABA content in droughted leaves of C. japonense (Y) and C. nankingense (N). C: Control (no PEG), T: PEG treatment. ** Value significant at P ≤ 0.01. Values given as mean ± SD (n = 3). SD's indicated by a bar.

    DISCUSSION

      The two Chrysanthemum spp. responded differentially to simulated drought stress

    • C. nankingense, the more drought sensitive of the two Chrysanthemum spp., developed signs of drought-induced damage earlier than C. japonense and the wilting index of the former was consistently higher at each time point (Fig. 1a). In agreement with this differential response, the RWC of C. japonense was greater than that of C. nankingense (Fig. 1b), supporting the use of RWC as an indirect means of classifying crop varieties for their drought sensitivity[41,42].

      The association between leaf surface features and drought tolerance

    • The leaf surfaces of the two species differs greatly. Leaf trichomes have been considered as a physical barrier against drought and high temperature stress[43]. They could increase water-use efficiency by increasing leaf boundary-layer resistance, thereby reducing transpirational water loss[44]. The more tolerant species developed a much higher density of trichomes on its leaves (Table 1). As this trait is readily visible, it would be attractive as an indirect selection criterion for improving drought tolerance[45]. Cuticular wax deposition represents an important mechanism for limiting non-stomatal water loss[46]. The quantity of cuticular wax on the surface of the leaves of C. japonense was markedly greater than on those of C. nankingense (Fig. 3a), consistent with their ranking with respect to drought tolerance. There were also significant differences between the two species with respect to the composition of cuticular wax (Fig. 3b), with the wax in the more tolerant species being richer in fatty alcohols and esters. Wax has been reported to affect the drought tolerance of plants in many species, among which, it has been reported the wax content of sunflower increased under drought condition[47]. To our knowledge, this is the first documented description of the composition of chrysanthemum cuticular wax.

      The association between antioxidant enzyme activity and drought tolerance

    • Drought stress is often accompanied by the accumulation of ROS, which induce oxidative stress[48]. Plants have evolved a number of means to scavenge ROS molecules, and the enzyme SOD is considered to be part of the first line of this defence[49]. SOD, CAT, APX, POD all reduces superoxide. The activity of all four of these enzymes was increased by drought stress in both species (Fig. 4), although SOD activity was enhanced more in C. japonense than in C. nankingense. Significant increases in the activity of both APX and CAT were observed in the early phase of the stress exposure, particularly in C. nankingense, while more modest increases were observed for C. japonense; enzyme activity remained higher in C. japonense than in C. nankingense after 6 h of stress. Experiments conducted in rice have similarly shown that the more tolerant cultivars tend to express higher levels of CAT and APX activity[50]. It has been suggested that in soybean[51] , sorghum[52] and sunflower[47], drought tolerance is associated with enhanced POD activity. However, this does not appear to apply to Chrysanthemum spp., since the level of POD activity was similar in both species after 10 h of stress (Fig. 4b). Drought stress induces extensive lipid peroxidation, allowing MDA (a by-product of lipid peroxidation) content to be exploited as an indicator of stress-induced oxidative damage to membranes[53,54]. Finally, EL provides a measure of cell integrity, and so has been frequently used as a surrogate for stress tolerance[55,56]. The levels of both EL and MDA in C. japonense were uniformly lower than in C. nankingense, at least over the first 10 h of stress treatment (Fig. 5), supporting the conclusion that C. japonense is a more drought tolerant species than C. nankingense.

      The association between osmotic regulation and drought tolerance

    • Plants take advantage of various molecules as osmoregulants, in particular the amino acid proline[57]. C. japonense with strong drought tolerance clearly accumulated more proline than C. nankingense when the plants were exposed to drought stress (Fig. 6), similar results were observed in other species including sunflower[47], rice[58] and Arabidopsis[24]. Proline contributes to the stabilization of sub-cellular structures, the scavenging of ROS and to buffering of cellular redox-potential under stress conditions[59]. The enhanced ability of the C. japonense leaf to accumulate proline thus may well provide a more favorable osmotic environment and a more stable cell membrane during episodes of drought stress.

      The association between photosynthesis and drought tolerance

    • Photosynthesis is very sensitive to drought stress. The photosynthetic parameters Pn, Gs and Tr were all significantly compromised in both Chrysanthemum spp. by drought stress (Fig. 7a−c). Zhang et al.[60] has similarly reported that moisture stressed Atractylodes lancea suffers a reduction in photosynthesis as measured by Gs and Pn. An early response to drought stress is stomatal closure, which serves to limit transpirational loss[61]. After 2 h of stress, C. nankingense had significant higher Tr than C. japonense (Fig. 7c), thus resulting in more water loss in leaves, which might explain faster loss in RWC of C. nankingense than that in C. japonense. Changes in Gs depend on leaf RWC[62], and Gs and Tr were both correlated with leaf RWC in both species. It is generally considered that drought-induced stomatal closure would certainly have suppressed photosynthesis[63,64]. Gs and Pn decreased rapidly in both species under PEG treatment (Fig. 7a, b). Under a more prolonged period of moisture deficiency, the leaf tissue becomes increasingly dehydrated, inducing metabolic impairment and a restriction in photophosphorylation capacity[62,65]. When stomatal conductance falls below a threshold of 50 mmol H2O m−2·s−1, limitations of non-stomatal processes become more important[66]. Here, Gs remained above this threshold in the first four hours of stress, but dropped below it by 6 h in C. nankingense but not in C. japonense (Fig. 7b), suggesting that the photosynthetic apparatus of C. nankingense suffered earlier and more severe damage. Ci increased slightly in both species under PEG stress (Fig. 7d), as also observed in cotton, vetiver grass and wheat[67−69]. An overestimate in Ci could arise from heterogeneous (or 'patch') stomatal closure and cuticular conductance, which have been identified as potential sources of error in the calculation of Ci in drought affected plants[70]. This may explain why Ci rose at a time when Gs and the RWC were low. Dark-adapted Fv/Fm values and estimates of chlorophyll content decreased in both species under PEG stress (Fig. 7e, f). A decline in PSII quantum efficiency during periods of stress has been noted in a number of plant species[71−73]. Low Fv/Fm ratios have been related to photoinhibition[74], since plants frequently absorb more light energy than they require for photosynthesis, particularly under drought conditions. Due to the limited reaction capacity of converting solar energy into chemical energy, excessive light absorption exacerbates the inactivation of PSII under drought, freeing electrons for the formation of ROS[75]. Both the Fv/Fm ratio and the chlorophyll content decreased more sharply for C. nankingense than for C. japonense. After 10 h of PEG stress, C. japonense leaves retained a higher chlorophyll content and a larger Fv/Fm ratio than those of C. nankingense (Fig. 7e, f), symptomatic of C. japonense being able to maintain a higher photosynthetic capacity under drought stress. Similarly, drought tolerant bean and edamame cultivars have been reported to retain a higher chlorophyll content and a superior Fv/Fm ratio than do more susceptible ones[76,77].

      The association between ABA content and drought tolerance

    • ABA, one of the most important metabolites produced under drought stress, is known to regulate plant water balance and drought stress tolerance[78]. Analysis of ABA-deficient mutants and -related genes have shown that this hormone is essential for triggering many of the important responses to drought stress[79]. Here, it was obvious that the ABA level in the leaf of both species was greatly enhanced by the imposition of drought stress (Fig. 8). The ABA content was significantly higher in C. japonense than in C. nankingense. In droughted-stressed durum wheat, Mahdid et al. have shown that a more tolerant cultivar accumulated more ABA than did a less tolerant one[80]. ABA is thought to increase hydraulic conductivity from the roots to the transpiring tissues[81], acting in conjunction with ABA-induced stomatal closure to restore a favorable water status to the leaf tissue. Gs and ABA appeared to be negatively correlated in both species. ABA may also influence osmotic regulation, ion and solute transport loading in growing cells, and so play a vital role in both water retention and protein and membrane protection[82]. Low water potential-induced proline accumulation in A. thaliana requires wild-type levels of ABA[83], while drought-induced changes in the synthesis of proline have been shown to be ABA dependent[84]. ABA plays a role in the upstream of proline accumulation by regulating the expression of key enzyme genes of proline biosynthesis, which also improves the adaptation of rice to hypoxia stress to a certain extent[85]. The present data indicate that the improved capacity to accumulate proline shown by C. japonense may be associated with its enhanced ability to accumulate ABA.

    CONCLUSIONS

    • Overall, it is clear that these two Chrysanthemum species show contrasting responses to drought stress at the morphological, physiological and biochemical levels. The superior tolerance of C. japonense likely flows from a combination of its better developed trichome layer, its higher cuticular wax content, its more rapid and abundant accumulation of ABA, its more flexible photosynthesis capacity, and its more effective osmoprotective and antioxidative system. The evaluation of the drought tolerance of the two chrysanthemum species further enriched the drought tolerance germplasm resource bank of chrysanthemum, clarified the different physiological and biochemical responses of two chrysanthemum species with great differences in drought tolerance, which has certain guiding significance for further development and application of drought tolerance resources of chrysanthemum.

      Acknowledgments

      • This study is supported by the National Natural Science Foundation of China (31870306), the National Key Research and Development Program of China (2020YFE0202900), the Fundamental Research Funds for Central Universities (KYZZ2022004).

      Conflict of interest

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

      • # These authors contributed equally: Yi Zhang, Jing Gu

      Rights and permissions
      • 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)  Table (1) References (85)
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    Cite this article
    Zhang Y, Gu J, Xia X, Zeng J, Sun H, et al. 2022. Contrasting responses to drought stress between Chrysanthemum japonense and C. nankingense. Ornamental Plant Research 2:16 doi: 10.48130/OPR-2022-0016
    Zhang Y, Gu J, Xia X, Zeng J, Sun H, et al. 2022. Contrasting responses to drought stress between Chrysanthemum japonense and C. nankingense. Ornamental Plant Research 2:16 doi: 10.48130/OPR-2022-0016
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Catalog

  • Html

  • Introduction
  • Materials and methods

  • Plant material and stress treatments

  • Water status

  • Characterization of the leaf surface and its cuticular wax

  • Enzyme assays

  • Cell membrane stability, malondialdehyde (mda) and free profine content

  • Chlorophyll content and photosynthesis related parameters

  • Endogenous aba level

  • Statistical analysis

  • Results

  • Plant drought stress phenotype and leaf water status

  • Leaf surface morphology

  • Cuticular wax amount and composition

  • Antioxidant enzyme activity

  • El and mda content

  • Free proline content

  • The effect of drought stress on photosynthetic parameters

  • Leaf aba content

  • Discussion

  • The two chrysanthemum spp. responded differentially to simulated drought stress

  • The association between leaf surface features and drought tolerance

  • The association between antioxidant enzyme activity and drought tolerance

  • The association between osmotic regulation and drought tolerance

  • The association between photosynthesis and drought tolerance

  • The association between aba content and drought tolerance

  • Conclusions
  • Acknowledgments
  • Conflict of interest
  • Rights and permissions
  • About this article
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