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2024 Volume 4
Article Contents
Abstract
Introduction
Methods
Bioinformatics analysis of the ARF18 gene
Plant materials and cultivation methods
Plasmid construction and plant transformation
Extracting plant genomic DNA and RNA
Real-Time quantitative Polymerase Chain Reaction (qPCR)
GUS staining
Determination of nitrate content, nitrate reductase activity
Yeast one-hybrid (Y1H) assay
Dual luciferase assays
Protein degradation assays in vitro
Data analysis
Results
Auxin regulates nitrogen uptake and utilization in apple
Transcript levels of MdARFs are induced by nitrate
Phylogenetic relationships and multiple sequence alignment of MdARF18
Overexpression of MdARF18 affects root development
Overexpression of MdARF18 inhibits nitrate uptake in plants
MdARF18 targets genes related to nitrogen uptake and utilization
MdARF18 binds to the promoter of MdNRT1.1 to restrain its expression
Discussion
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
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ARTICLE   Open Access    

Identification and content analysis of volatile components in 100 cultivars of Chinese herbaceous peony

  • 1.

    College of Agriculture/Tree Peony, Henan University of Science and Technology, Luoyang 471000, China

  • 2.

    Luoyang Academy of Agriculture and Forestry Sciences, Luoyang 471000, China

  • # Authors contributed equally: Aixin Wang, Yasang Luo

More Information
  • Herbaceous peony (Paeonia lactiflora Pall.) is a well-known and traditional flower in China, occupying a significant position in Chinese traditional culture. The floral scent of the herbaceous peony however remains relatively understudied. The objective of this study was to investigate the floral composition of herbaceous peony by collecting and identifying floral volatiles from 100 cultivars, including P. lactiflora 'Hangbaishao', P. lactiflora 'Hongrongqiu', P. lactiflora 'Biandihong', P. lactiflora 'Zijin Daipao', P. lactiflora 'Zixia Yingxue', and P. lactiflora 'Fenchi Dicui'. The volatile compounds were collected using the dynamic headspace technique and identified through gas chromatography-mass spectrometry (GC-MS). The results demonstrated qualitative and quantitative variations in the floral fragrances emitted by the 100 cultivars, with a total of 16 volatiles belonging to six categories (six alkanes, three alcohols and esters, two terpenes, as well as one each of ether and phenol) being identified. However, it is notable that not all volatile categories were emitted by every cultivar. Moreover, while some compounds were present in all 100 herbaceous peony cultivars, others were exclusive to specific cultivars. The screening revealed that ten of the 16 identified flower volatile compounds exhibited unique floral components. It is noteworthy that benzene,1,4-dimethoxy-, was identified as the most prominent compound in several cultivars, including P. lactiflora 'Taohua Huancai', P. lactiflora 'Xishifen', P. lactiflora 'Dabanhong', P. lactiflora 'Fumantang', and P. lactiflora 'Zhushapan'. Furthermore, the clustering classification results demonstrated that benzene,1,4-dimethoxy-, exhibited the highest variable importance in projection (VIP) value of 3.153, as determined by partial least squares discriminant analysis (PLS-DA).

  • Introduction

    Crops require a variety of nutrients for growth and nitrogen is particularly important. Nitrogen is the primary factor limiting plant growth and yield formation, and it also plays a significant role in improving product quality[1−4]. Nitrogen accounts for 1%−3% of the dry weight of plants and is a component of many compounds. For example, it is an important part of proteins, a component of nucleic acids, the skeleton of cell membranes, and a constituent of chlorophyll[5,6]. When the plant is deficient in nitrogen, the synthesis process of nitrogen-containing substances such as proteins decrease significantly, cell division and elongation are restricted, and chlorophyll content decreases, and this leads to short and thin plants, small leaves, and pale color[2,7,8]. If nitrogen in the plant is in excess, a large number of carbohydrates will be used for the synthesis of proteins, chlorophyll, and other substances, so that cells are large and thin-walled, and easy to be attacked by pests and diseases. At the same time, the mechanical tissues in the stem are not well developed and are prone to collapse[3,8,9]. Therefore, the development of new crop varieties with both high yields and improved nitrogen use efficiency (NUE) is an urgently needed goal for more sustainable agriculture with minimal nitrogen demand.

    Plants obtain inorganic nitrogen from the soil, mainly in the form of NH4+ and nitrate (NO3−)[10−13]. Nitrate uptake by plants occurs primarily in aerobic environments[3]. Transmembrane proteins are required for nitrate uptake from the external environment as well as for transport and translocation between cells, tissues, and organs. NITRATE TRANSPORTER PROTEIN 1 (NRT1)/PEPTIDE TRANSPORTER (PTR) family (NPF), NRT2, CHLORIDE CHANNEL (CLC) family, and SLOW ACTIVATING ANION CHANNEL are four protein families involved in nitrate transport[14]. One of the most studied of these is NRT1.1, which has multiple functions[14]. NRT1.1 is a major nitrate sensor, regulating many aspects of nitrate physiology and developmental responses, including regulating the expression levels of nitrate-related genes, modulating root architecture, and alleviating seed dormancy[15−18].

    There is mounting evidence that plant growth and development are influenced by interactions across numerous phytohormone signaling pathways, including abscisic acid, gibberellins, growth hormones, and cytokinins[3,19,20]. To increase the effectiveness of plant nitrogen fertilizer application, it may be possible to tweak the signaling mediators or vary the content of certain phytohormones. Since the 1930s, research on the interplay between growth factors and N metabolism has also been conducted[3]. The Indole acetic acid (IAA) level of plant shoots is shown to decrease in early studies due to N shortage, although roots exhibit the reverse tendency[3,21]. In particular, low NO3− levels caused IAA buildup in the roots of Arabidopsis, Glycine max, Triticum aestivum, and Zea mays, indicating that IAA is crucial for conveying the effectiveness of exogenous nitrogen to the root growth response[20,22,23].

    Studies have shown that two families are required to control the expression of auxin-responsive genes: one is the Auxin Response Factor (ARF) and the other is the Aux/IAA repressor family[24−26]. As the transcription factor, the ARF protein regulates the expression of auxin response genes by specifically binding to the TGTCNN auxin response element (AuxRE) in promoters of primary or early auxin response genes[27]. Among them, rice OsARF18, as a class of transcriptional repressor, has been involved in the field of nitrogen utilization and yield[23,28]. In rice (Oryza sativa), mutations in rice salt tolerant 1 (rst1), encoding the OsARF18 gene, lead to the loss of its transcriptional repressor activity and up-regulation of OsAS1 expression, which accelerates the assimilation of NH4+ to Asn and thus increases N utilization[28]. In addition, dao mutant plants deterred the conversion of IAA to OxIAA, thus high levels of IAA strongly activates OsARF18, which subsequently represses the expression of OsARF2 and OsSUT1 by directly binding to the AuxRE and SuRE promoter motifs, resulting in the inhibition of carbohydrate partitioning[23]. As a result, rice carrying the dao has low yields.

    Apples (Malus domestica) are used as a commercially important crop because of their high ecological adaptability, high nutritional value, and annual availability of fruit[29]. To ensure high apple yields, growers promote rapid early fruit yield growth by applying nitrogen. However, the over-application of nitrogen fertilizer to apples during cultivation also produces common diseases and the over-application of nitrogen fertilizer is not only a waste of resources but also harmful to the environment[29]. Therefore, it is of great significance to explore efficient nitrogen-regulated genes to understand the uptake and regulation of nitrogen fertilizer in apples, and to provide reasonable guidance for nitrogen application during apple production[30]. In this study, MdARF18 is identified which is a key transcription factor involved in nitrate uptake and transport in apples and MdARF18 reduces NO3− uptake and assimilation. Further analysis suggests that MdRF18 may inhibit the transcriptional level of MdNRT1.1 promoter by directly binding to its TGTCTT target, thus affecting normal plant growth.

    Methods

    Bioinformatics analysis of the ARF18 gene

    The protein sequence of apple MdARF18 (MD07G1152100) was obtained from The Apple Genome (https://iris.angers.inra.fr/gddh13/). Mutant of arf18 (GABI_699B09) sequence numbers were obtained from the official TAIR website (www.arabidopsis.org). The protein sequences of ARF18 from different species were obtained from the protein sequence of apple MdARF18 on the NCBI website. Using these data, a phylogenetic tree with reasonably close associations was constructed[31].

    Protein structural domain prediction of ARF18 was performed on the SMART website (https://smart.embl.de/). Motif analysis of ARF18 was performed by MEME (https://meme-suite.org/meme/tools/meme). Clustal was used to do multiple sequence comparisons. The first step was accessing the EBI web server through the Clustal Omega channel. The visualization of the results was altered using Jalview, which may be downloaded from www.jalview.org/download.[32]

    Plant materials and cultivation methods

    The apple 'Orin' callus was transplanted on MS medium containing 1.5 mg·L−1 6-benzylaminopurine (6-BA) and 0.5 mg·L−1 2,4 dichlorophenoxyacetic acid (2,4-D) at 25 °C, in the dark, at 21-d intervals. 'Royal Gala' apple cultivars were cultured in vermiculite and transplanted at 25 °C every 30 d. The Arabidopsis plants used were of the Columbia (Col-0) wild-type variety. Sowing and germinating Arabidopsis seeds on MS nutrient medium, and Arabidopsis seeds were incubated and grown at 25 °C (light/dark cycle of 16 h/8 h)[33].

    The nutrient solution in the base contained 1.0 mM CaCl2, 1.0 mM KH2PO4, 1.0 mM MgSO4, 0.1 mM FeSO4·7H2O 0.1 mM Na2EDTA·2H2O, 50 μM MnSO4·H2O, 50 μM H3BO3, 0.05 μM CuSO4·5H2O, 0.5 μM Na2MoO4·2H2O, 15 μM ZnSO4·7H2O, 2.5 μM KI, and 0.05 μM CoCl·6H2O, and 0.05 μM CoCl·6H2O, and 0.05 μM CoCl· 6H2O. 2H2O, 15 μM ZnSO4·7H2O, 2.5 μM KI and 0.05 μM CoCl·6H2O, and 0.05 μM CoCl·6H2O, supplemented with 0.5 mM, 2 mM, and 10 mM KNO3 as the sole nitrogen source, and added with the relevant concentrations of KCl to maintain the same K concentration[33,34].

    For auxin treatment, 12 uniformly growing apple tissue-cultured seedlings (Malus domestica 'Royal Gala') were selected from each of the control and treatment groups, apple seedlings were incubated in a nutrient solution containing 1.5 mg·L−1 6-BA, 0.2 mg·L−1 naphthalene acetic acid, and IAA (10 μM) for 50 d, and then the physiological data were determined. Apple seedlings were incubated and grown at 25 °C (light/dark cycle of 16 h/8 h).

    For nitrate treatment, Arabidopsis seedlings were transferred into an MS medium (containing different concentrations of KNO3) as soon as they germinated to test root development. Seven-day-old Arabidopsis were transplanted into vermiculite and then treated with a nutrient solution containing different concentrations of KNO3 (0.5, 2, 10 mM) and watered at 10-d intervals. Apple calli were treated with medium containing 1.5 mg·L−1 6-BA, 0.5 mg·L−1 2,4-D, and varying doses of KNO3 (0.5, 2, and 10 mM) for 25 d, and samples were examined for relevant physiological data. Apple calli were subjected to the same treatment for 1 d for GUS staining[35].

    Plasmid construction and plant transformation

    To obtain MdARF18 overexpression materials, the open reading frame (ORF) of MdARF18 was introduced into the pRI-101 vector. To obtain pMdNRT1.1 material, the 2 kb segment located before the transcription start site of MdNRT1.1 was inserted into the pCAMBIA1300 vector. The Agrobacterium tumefaciens LBA4404 strain was cultivated in lysozyme broth (LB) medium supplemented with 50 mg·L−1 kanamycin and 50 mg·L−1 rifampicin. The MdARF18 overexpression vector and the ProMdNRT1.1::GUS vector were introduced into Arabidopsis and apple callus using the flower dip transformation procedure. The third-generation homozygous transgenic Arabidopsis (T3) and transgenic calli were obtained[36]. Information on the relevant primers designed is shown in Supplemental Table S1.

    Extracting plant genomic DNA and RNA

    Plant DNA and RNA were obtained using the Genomic DNA Kit and the Omni Plant RNA Kit (tDNase I) (Tiangen, Beijing, China)[37].

    Real-Time quantitative Polymerase Chain Reaction (qPCR)

    cDNA was synthesized for qPCR by using the PrimeScript First Strand cDNA Synthesis Kit (Takara, Dalian, China). The cDNA for qPCR was synthesized by using the PrimeScript First Strand cDNA Synthesis Kit (Takara, Dalian, China). Quantitative real-time fluorescence analysis was performed by using the UltraSYBR Mixture (Low Rox) kit (ComWin Biotech Co. Ltd., Beijing, China). qRT-PCR experiments were performed using the 2−ΔΔCᴛ method for data analysis. The data were analyzed by the 2−ΔΔCᴛ method[31].

    GUS staining

    GUS staining buffer contained 1 mM 5-bromo-4-chloro-3-indolyl-β-glutamic acid, 0.01 mM EDTA, 0.5 mM hydrogen ferrocyanide, 100 mM sodium phosphate (pH 7.0), and 0.1% (v/v) Triton X-100 was maintained at 37 °C in the dark. The pMdNRT1.1::GUS construct was transiently introduced into apple calli. To confirm whether MdNRT1.1 is activated or inhibited by MdARF18, we co-transformed 35S::MdARF18 into pMdNRT1.1::GUS is calling. The activity of transgenic calli was assessed using GUS labeling and activity assays[33,38].

    Determination of nitrate content, nitrate reductase activity

    The specimens were crushed into fine particles, combined with 1 mL of ddH2O, and thereafter subjected to a temperature of 100 °C for 30 min. The supernatant was collected in a flow cell after centrifugation at 12,000 revolutions per minute for 10 min. The AutoAnalyzer 3 continuous flow analyzer was utilized to measure nitrate concentrations. (SEAL analytical, Mequon, WI, USA). Nitrate reductase (NR) activity was characterized by the corresponding kits (Solarbio Life Science, Beijing, China) using a spectrophotometric method[31].

    Yeast one-hybrid (Y1H) assay

    Y1H assays were performed as previously described by Liu et al.[39]. The coding sequence of MdARF18 was integrated into the pGADT7 expression vector, whereas the promoter region of MdNRT1.1 was included in the pHIS2 reporter vector. Subsequently, the constitutive vectors were co-transformed into the yeast monohybrid strain Y187. The individual transformants were assessed on a medium lacking tryptophan, leucine, and histidine (SDT/-L/-H). Subsequently, the positive yeast cells were identified using polymerase chain reaction (PCR). The yeast strain cells were diluted at dilution factors of 10, 100, 1,000, and 10,000. Ten μL of various doses were added to selective medium (SD-T/-L/-H) containing 120 mM 3-aminotriazole (3-AT) and incubated at 28 °C for 2−3 d[37].

    Dual luciferase assays

    Dual-luciferase assays were performed as described previously[40]. Full-length MdARF18 was cloned into pGreenII 62-SK to produce MdARF18-62-SK. The promoter fragment of MdNRT1.1 was cloned into pGreenII 0800-LUC to produce pMdNRT1.1-LUC. Different combinations were transformed into Agrobacterium tumefaciens LBA4404 and the Agrobacterium solution was injected onto the underside of the leaves of tobacco (Nicotiana benthamiana) leaves abaxially. The Dual Luciferase Reporter Kit (Promega, www.promega.com) was used to detect fluorescence activity.

    Protein degradation assays in vitro

    Total protein was extracted from wild-type and transgenic apple calli with or without 100 μM MG132 treatment. The purified MdARF18-HIS fusion protein was incubated with total protein[41]. Samples were collected at the indicated times (0, 1, 3, 5, and 7 h).

    Protein gel blots were analyzed using GST antibody. ACTIN antibody was used as an internal reference. All antibodies used in this study were provided by Abmart (www.ab-mart.com).

    Data analysis

    Unless otherwise noted, every experiment was carried out independently in triplicate. A one-way analysis of variance (ANOVA) was used to establish the statistical significance of all data, and Duncan's test was used to compare results at the p < 0.05 level[31].

    Results

    Auxin regulates nitrogen uptake and utilization in apple

    To investigate whether auxin affects the effective uptake of nitrate in apple, we first externally applied IAA under normal N (5 mM NO3−) environment, and this result showed that the growth of Gala apple seedlings in the IAA-treated group were better than the control, and their fresh weights were heavier than the control group (Fig. 1a, d). The N-related physiological indexes of apple seedlings also showed that the nitrate content and NR activity of the root part of the IAA-treated group were significantly higher than the control group, while the nitrate content and NR activity of the shoot part were lower than the control group (Fig. 1b, c). These results demonstrate that auxin could promote the uptake of nitrate and thus promotes growth of plants.

    Figure 1.  Auxin enhances nitrate uptake of Gala seedlings. (a) Phenotypes of apple (Malus domestica 'Royal Gala') seedlings grown nutritionally for 50 d under IAA (10 μM) treatment. (b) Nitrate content of shoot and root apple (Malus domestica 'Royal Gala') seedlings treated with IAA. (c) NR activity in shoot and root of IAA treatment apple (Malus domestica 'Royal Gala') seedlings. (d) Seedling fresh weight under IAA treatment. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    To test whether auxin affects the expression of genes related to nitrogen uptake and metabolism. For the root, the expression levels of MdNRT1.1, MdNRT2.1, MdNIA1, MdNIA2, and MdNIR were higher than control group (Supplemental Fig. S1a, f, h−j), while the expression levels of MdNRT1.2, MdNRT1.6 and MdNRT2.5 were lower than control group significantly (Supplemental Fig. S1b, d, g). For the shoot, the expression of MdNRT1.1, MdNRT1.5, MdNRT1.6, MdNRT1.7, MdNRT2.1, MdNRT2.5, MdNIA1, MdNIA2, and MdNIR genes were significantly down-regulated (Supplemental Fig. S1a, c−j). This result infers that the application of auxin could mediate nitrate uptake in plants by affecting the expression levels of relevant nitrate uptake and assimilation genes.

    Transcript levels of MdARFs are induced by nitrate

    Since the auxin signaling pathway requires the regulation of the auxin response factors (ARFs)[25,27], it was investigated whether members of ARF genes were nitrate responsive. Firstly, qPCR quantitative analysis showed that the five subfamily genes of MdARFs (MdARF9, MdARF2, MdARF12, MdARF3, and MdARF18) were expressed at different levels in various organs of the plant (Supplemental Fig. S2). Afterward, the expression levels of five ARF genes were analyzed under different concentrations of nitrate treatment (Fig. 2), and it was concluded that these genes represented by each subfamily responded in different degrees, but the expression level of MdARF18 was up-regulated regardless of low or high nitrogen (Fig. 2i, j), and the expression level of MdARF18 showed a trend of stable up-regulation under IAA treatment (Supplemental Fig. S3). The result demonstrates that MdARFs could affect the uptake of external nitrate by plants and MdARF18 may play an important role in the regulation of nitrate uptake.

    Figure 2.  Relative expression analysis of MdARFs subfamilies in response to different concentrations of nitrate. Expression analysis of representative genes from five subfamilies of MdARF transcription factors. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    Phylogenetic relationships and multiple sequence alignment of MdARF18

    MdARF18 (MD07G1152100) was predicted through The Apple Genome website (https://iris.angers.inra.fr/gddh13/) and it had high fitness with AtARF18 (AT3G61830). The homologs of ARF18 from 15 species were then identified in NCBI (www.ncbi.nlm.nih.gov) and then constructed an evolutionary tree (Supplemental Fig. S4). The data indicates that MdARF18 was most closely genetically related to MbARF18 (Malus baccata), indicating that they diverged recently in evolution (Supplemental Fig. S4). Conserved structural domain analyses indicated that all 15 ARF18 proteins had highly similar conserved structural domains (Supplemental Fig. S5). In addition, multiple sequence alignment analysis showed that all 15 ARF18 genes have B3-type DNA-binding domains (Supplemental Fig. S6), which is in accordance with the previous reports on ARF18 protein structure[26].

    Overexpression of MdARF18 affects root development

    To explore whether MdARF18 could affect the development of the plant's root system. Firstly, MdARF18 was heterologously expressed into Arabidopsis, and an arf18 mutant (GABI_699B09) Arabidopsis was also obtained (Supplemental Fig. S7). Seven-day-old MdARF18 transgenic Arabidopsis and arf18 mutants were treated in a medium with different nitrate concentrations for 10 d (Fig. 3a, b). After observing results, it was found that under the environment of high nitrate concentration, the primary root of MdARF18 was shorter than arf18 and wild type (Fig. 3c), and the primary root length of arf18 is the longest (Fig. 3c), while there was no significant difference in the lateral root (Fig. 3d). For low nitrate concentration, there was no significant difference in the length of the primary root, and the number of lateral roots of MdARF18 was slightly more than wild type and arf18 mutant. These results suggest that MdARF18 affects root development in plants. However, in general, low nitrate concentrations could promote the transport of IAA by NRT1.1 and thus inhibit lateral root production[3], so it might be hypothesized that MdARF18 would have some effect on MdNRT1.1 thus leading to the disruption of lateral root development.

    Figure 3.  MdARF18 inhibits root development. (a) MdARF18 inhibits root length at 10 mM nitrate concentration. (b) MdARF18 promotes lateral root growth at 0.5 mM nitrate concentration. (c) Primary root length statistics. (d) Lateral root number statistics. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).
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    Overexpression of MdARF18 inhibits nitrate uptake in plants

    To investigate whether MdARF18 affects the growth of individual plants under different concentrations of nitrate, 7-day-old overexpression MdARF18, and arf18 mutants were planted in the soil and incubated for 20 d. It was found that arf18 had the best growth of shoot, while MdARF18 had the weakest shoot growth at any nitrate concentration (Fig. 4a). MdARF18 had the lightest fresh weight and the arf18 mutant had the heaviest fresh weight (Fig. 4b). N-related physiological indexes revealed that the nitrate content and NR activity of arf18 were significantly higher than wild type, whereas MdARF18 materials were lower than wild type (Fig. 4c, d). More detail, MdARF18 had the lightest fresh weight under low and normal nitrate, while the arf18 mutant had the heaviest fresh weight, and the fresh weight of arf18 under high nitrate concentration did not differ much from the wild type (Fig. 4b). Nitrogen-related physiological indexes showed that the nitrate content of arf18 was significantly higher than wild type, while MdARF18 was lower than wild type. The NR activity of arf18 under high nitrate did not differ much from the wild type, but the NR activity of MdARF18 was the lowest in any treatment (Fig. 4c, d). These results indicate that MdARF18 significantly inhibits plant growth by inhibiting plants to absorb nitrate, and is particularly pronounced at high nitrate concentrations.

    Figure 4.  Ectopic expression of MdARF18 inhibits Arabidopsis growth. (a) Status of Arabidopsis growth after one month of incubation at different nitrate concentrations. (b) Fresh weight of Arabidopsis. (c) Nitrate content of Arabidopsis. (d) NR activity in Arabidopsis. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    In addition, to further validate this conclusion, MdARF18 overexpression calli were obtained and treated with different concentrations of nitrate (Supplemental Fig. S8). The results show that the growth of overexpressed MdARF18 was weaker than wild type in both treatments (Supplemental Fig. S9a). The fresh weight of MdARF18 was significantly lighter than wild type (Supplemental Fig. S9b), and its nitrate and NR activity were lower than wild type (Supplemental Fig. S9c, d), which was consistent with the above results (Fig. 4). This result further confirms that MdARF18 could inhibit the development of individual plants by inhibiting the uptake of nitrate.

    MdARF18 targets genes related to nitrogen uptake and utilization

    Nitrate acts as a signaling molecule that takes up nitrate by activating the NRT family as well as NIAs and NIR[3,34]. To further investigate the pathway by which MdARF18 inhibits plant growth and reduces nitrate content, qRT-PCR was performed on the above plant materials treated with different concentrations of nitrate (Fig. 5). The result shows that the expression levels of AtNRT1.1, AtNIA1, AtNIA2, and AtNIR were all down-regulated in overexpression of MdARF18, and up-regulated in the arf18 mutant (Fig. 5a, h−j). There was no significant change in AtNRT1.2 at normal nitrate levels, but AtNRT1.2 expression levels were down-regulated in MdARF18 and up-regulated in arf18 at both high and low nitrate levels (Fig. 5b). This trend in the expression levels of these genes might be consistent with the fact that MdARF18 inhibits the expression of nitrogen-related genes and restricts plant growth. The trend in the expression levels of these genes is consistent with MdARF18 restricting plant growth by inhibiting the expression of nitrogen-related genes. However, AtNRT1.5, AtNRT1.6, AtNRT1.7, AtNRT2.1, and AtNRT2.5 did not show suppressed expression levels in MdARF18 (Fig. 5c−g). These results suggest that MdARF18 inhibits nitrate uptake and plant growth by repressing some of the genes for nitrate uptake or assimilation.

    Figure 5.  qPCR-RT analysis of N-related genes. Expression analysis of N-related genes in MdARF18 transgenic Arabidopsis at different nitrate concentrations. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    In addition, to test whether different concentrations of nitrate affect the protein stability of MdARF18. However, it was found that there was no significant difference in the protein stability of MdARF18 at different concentrations of nitrate (Supplemental Fig. S10). This result suggests that nitrate does not affect the degradation of MdARF18 protein.

    MdARF18 binds to the promoter of MdNRT1.1 to restrain its expression

    To further verify whether MdARF18 can directly bind N-related genes, firstly we found that the MdNRT1.1 promoter contains binding sites to ARF factors (Fig. 6a). The yeast one-hybrid research demonstrated an interaction between MdARF18 and the MdNRT1.1 promoter, as shown in Fig. 6b. Yeast cells that were simultaneously transformed with MdNRT1.1-P-pHIS and pGADT7 were unable to grow in selected SD medium. However, cells that were transformed with MdNRT1.1-P-pHIS and MdARF18-pGADT7 grew successfully in the selective medium. The result therefore hypothesizes that MdARF18 could bind specifically to MdNRT1.1 promoter to regulate nitrate uptake in plants.

    Figure 6.  MdARF18 binds directly to the promoter of MdNRT1.1. (a) Schematic representation of MdNRT1.1 promoter. (b) Y1H assay of MdARF18 bound to the MdNRT1.1 promoter in vitro. 10−1, 10−2, 10−3, and 10−4 indicate that the yeast concentration was diluted 10, 100, 1,000, and 10,000 times, respectively. 3-AT stands for 3-Amino-1,2,4-triazole. (c) Dual luciferase assays demonstrate the binding of MdARF18 with MdNRT1.1 promoter. The horizontal bar on the left side of the right indicates the captured signal intensity. Empty LUC and 35S vectors were used as controls. Representative images of three independent experiments are shown here.
    DownLoad: Full-Size Img PowerPoint

    To identify the inhibition or activation of MdNRT1.1 by MdARF18, we analyzed their connections by Dual luciferase assays (Fig. 6c), and also analyzed the fluorescence intensity (Supplemental Fig. S11). It was concluded that the fluorescence signals of cells carrying 35Spro and MdNRT1.1pro::LUC were stronger, but the mixture of 35Spro::MdARF18 and MdNRT1.1pro::LUC injected with fluorescence signal intensity was significantly weakened. Next, we transiently transformed the 35S::MdARF18 into pMdNRT1.1::GUS transgenic calli (Fig. 7). GUS results first showed that the color depth of pMdNRT1.1::GUS and 35S::MdARF18 were significantly lighter than pMdnNRT1.1::GUS alone (Fig. 7a). GUS enzyme activity, as well as GUS expression, also indicated that the calli containing pMdnNRT1.1::GUS alone had a stronger GUS activity (Fig. 7b, c). In addition, the GUS activity of calli containing both pMdNRT1.1:GUS and 35S::MdARF18 were further attenuated under both high and low nitrate concentrations (Fig. 7a). These results suggest that MdARF18 represses MdNRT1.1 expression by directly binding to the MdNRT1.1 promoter region.

    Figure 7.  MdARF18 inhibits the expression of MdNRT1.1. (a) GUS staining experiment of pMdNRT1.1::GUS transgenic calli and transgenic calli containing both pMdNRT1.1::GUS and 35S::MdARF18 with different nitrate treatments. (b) GUS activity assays in MdARF18 overexpressing calli with different nitrate treatments. (c) GUS expression level in MdARF18 overexpressing calli with different nitrate treatments. Bars represent the mean ± SD (n = 3). Different numbers of asterisk above the bars indicate significant differences using the LSD test (*p < 0.05 and **p< 0.01).
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    Discussion

    Plants replenish their nutrients by absorbing nitrates from the soil[42,43]. Previous studies have shown that some of the plant hormones such as IAA, GA, and ABA interact with nitrate[25,44−45]. The effect of nitrate on the content and transport of IAA has been reported in previous studies, e.g., nitrate supply reduced IAA content in Arabidopsis, wheat, and maize roots and inhibited the transport of IAA from shoot to root[20,21]. In this study, it was found that auxin treatment promoted individual fresh weight gain and growth (Fig. 1a, b). Nitrate content and NR activity were also significantly higher in their root parts (Fig. 1c, d) and also affected the transcript expression levels of related nitrate uptake and assimilation genes (Supplemental Fig. S1). Possibly because IAA can affect plant growth by influencing the uptake of external nitrates by the plant.

    ARFs are key transcription factors to regulate auxin signaling[46−49]. We identified five representative genes of the apple MdARFs subfamily and they all had different expression patterns (Supplemental Fig. S2). The transcript levels of each gene were found to be affected to different degrees under different concentrations of nitrate, but the expression level of MdARF18 was up-regulated under both low and high nitrate conditions (Fig. 2). The transcript level of MdARF18 was also activated under IAA treatment (Supplemental Fig. S3), so MdARF18 began to be used in the study of the mechanism of nitrate uptake in plants. In this study, an Arabidopsis AtARF18 homolog was successfully cloned and named MdARF18 (Supplemental Figs S4, S5). It contains a B3-type DNA-binding structural domain consistent with previous studies of ARFs (Supplemental Fig. S6), and arf18 mutants were also obtained and their transcript levels were examined (Supplemental Fig. S7).

    Plants rely on rapid modification of the root system to efficiently access effective nitrogen resources in the soil for growth and survival. The plasticity of root development is an effective strategy for accessing nitrate, and appropriate concentrations of IAA can promote the development of lateral roots[7,44]. The present study found that the length of the primary root was shortened and the number of lateral roots did increase in IAA-treated Gl3 apple seedlings (Supplemental Fig. S12). Generally, an environment with low concentrations of nitrate promotes the transport of IAA by AtNRT1.1, which inhibits the growth of lateral roots[14]. However, in the research of MdARF18 transgenic Arabidopsis, it was found that the lateral roots of MdARF18-OX increased under low concentrations of nitrate, but there was no significant change in the mutant arf18 (Fig. 3). Therefore, it was hypothesized that MdARF18 might repress the expression of the MdNRT1.1 gene or other related genes that can regulate root plasticity, thereby affecting nitrate uptake in plants.

    In rice, several researchers have demonstrated that OsARF18 significantly regulates nitrogen utilization. Loss of function of the Rice Salt Tolerant 1 (RST1) gene (encoding OsARF18) removes its ability to transcriptionally repress OsAS1, accelerating the assimilation of NH4+ to Asn and thereby increasing nitrogen utilization[28]. During soil incubation of MdARF18-OX Arabidopsis, it was found that leaving aside the effect of differences in nitrate concentration, the arf18 mutant grew significantly better than MdARF18-OX and had higher levels of nitrate and NR activity in arf18 than in MdARF18-OX. This demonstrates that MdARF18 may act as a repressor of nitrate uptake and assimilation, thereby inhibiting normal plant development (Fig. 4). Interestingly, an adequate nitrogen environment promotes plant growth, but MdARF18-OX Arabidopsis growth and all physiological indexes were poorer under high nitrate concentration than MdARF18-OX at other concentrations. We hypothesize that MdARF18 may be activated more intensively at high nitrate concentrations, or that MdARF18 suppresses the expression levels of genes for nitrate uptake or assimilation (genes that may play a stronger role at high nitrate concentrations), thereby inhibiting plant growth. In addition, we obtained MdARF18 transgenic calli (Supplemental Fig. S8) and subjected them to high and low concentrations of nitrate, and also found that MdARF18 inhibited the growth of individuals at both concentrations (Supplemental Fig. S9). This further confirms that MdARF18 inhibits nitrate uptake in individuals.

    ARF family transcription factors play a key role in transmitting auxin signals to alter plant growth and development, e.g. osarf1 and osarf24 mutants have reduced levels of OsNRT1.1B, OsNRT2.3a and OsNIA2 transcripts[22]. Therefore, further studies are needed to determine whether MdARF18 activates nitrate uptake through different molecular mechanisms. The result revealed that the transcript levels of AtNRT1.1, AtNIA1, AtNIA2, and AtNIR in MdARF18-OX were consistent with the developmental pattern of impaired plant growth (Fig. 5). Unfortunately, we attempted to explore whether variability in nitrate concentration affects MdARF18 to differ at the protein level, but the two did not appear to differ significantly (Supplemental Fig. S10).

    ARF transcription factors act as trans-activators/repressors of N metabolism-related genes by directly binding to TGTCNN/NNGACA-containing fragments in the promoter regions of downstream target genes[27,50]. The NRT family plays important roles in nitrate uptake, transport, and storage, and NRT1.1 is an important dual-affinity nitrate transporter protein[7,50−52], and nitrogen utilization is very important for apple growth[53,54]. We identified binding sites in the promoters of these N-related genes that are compatible with ARF factors, and MdARF18 was found to bind to MdNRT1.1 promoter by yeast one-hybrid technique (Fig. 6a, b). It was also verified by Dual luciferase assays that MdARF18 could act as a transcriptional repressor that inhibited the expression of the downstream gene MdNRT1.1 (Fig. 6c), which inhibited the uptake of nitrate in plants. In addition, the GUS assay was synchronized to verify that transiently expressed pMdNRT1.1::GUS calli with 35S::MdARF18 showed a lighter staining depth and a significant decrease in GUS transcript level and enzyme activity (Fig. 7). This phenomenon was particularly pronounced at high concentrations of nitrate. These results suggest that MdARF18 may directly bind to the MdNRT1.1 promoter and inhibit its expression, thereby suppressing NO3− metabolism and decreasing the efficiency of nitrate uptake more significantly under high nitrate concentrations.

    Conclusions

    In conclusion, in this study, we found that MdARF18 responds to nitrate and could directly bind to the TGTCTT site of the MdNRT1.1 promoter to repress its expression. Our findings provide new insights into the molecular mechanisms by which MdARF18 regulates nitrate transport in apple.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and design: Liu GD; data collection: Liu GD, Rui L, Liu RX; analysis and interpretation of results: Liu GD, Li HL, An XH; draft manuscript preparation: Liu GD; supervision: Zhang S, Zhang ZL; funding acquisition: You CX, Wang XF; All authors reviewed the results and approved the final version of the manuscript.

    Data availability

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

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (32272683), the Shandong Province Key R&D Program of China (2022TZXD008-02), the China Agriculture Research System of MOF and MARA (CARS-27), the National Key Research and Development Program of China (2022YFD1201700), and the National Natural Science Foundation of China (NSFC) (32172538).

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

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  • Cite this article

    Wang A, Luo Y, Niu T, Gao K, Wang S, et al. 2024. Identification and content analysis of volatile components in 100 cultivars of Chinese herbaceous peony. Ornamental Plant Research 4: e032 doi: 10.48130/opr-0024-0029
    Wang A, Luo Y, Niu T, Gao K, Wang S, et al. 2024. Identification and content analysis of volatile components in 100 cultivars of Chinese herbaceous peony. Ornamental Plant Research 4: e032 doi: 10.48130/opr-0024-0029
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Identification and content analysis of volatile components in 100 cultivars of Chinese herbaceous peony

  • 1.

    College of Agriculture/Tree Peony, Henan University of Science and Technology, Luoyang 471000, China

  • 2.

    Luoyang Academy of Agriculture and Forestry Sciences, Luoyang 471000, China

  • Received: 02 July 2024
  • Revised: 11 September 2024
  • Accepted: 12 October 2024
  • Published online: 11 December 2024
Ornamental Plant Research  4,  Article number: e032  (2024)  |  Cite this article

Abstract: Herbaceous peony (Paeonia lactiflora Pall.) is a well-known and traditional flower in China, occupying a significant position in Chinese traditional culture. The floral scent of the herbaceous peony however remains relatively understudied. The objective of this study was to investigate the floral composition of herbaceous peony by collecting and identifying floral volatiles from 100 cultivars, including P. lactiflora 'Hangbaishao', P. lactiflora 'Hongrongqiu', P. lactiflora 'Biandihong', P. lactiflora 'Zijin Daipao', P. lactiflora 'Zixia Yingxue', and P. lactiflora 'Fenchi Dicui'. The volatile compounds were collected using the dynamic headspace technique and identified through gas chromatography-mass spectrometry (GC-MS). The results demonstrated qualitative and quantitative variations in the floral fragrances emitted by the 100 cultivars, with a total of 16 volatiles belonging to six categories (six alkanes, three alcohols and esters, two terpenes, as well as one each of ether and phenol) being identified. However, it is notable that not all volatile categories were emitted by every cultivar. Moreover, while some compounds were present in all 100 herbaceous peony cultivars, others were exclusive to specific cultivars. The screening revealed that ten of the 16 identified flower volatile compounds exhibited unique floral components. It is noteworthy that benzene,1,4-dimethoxy-, was identified as the most prominent compound in several cultivars, including P. lactiflora 'Taohua Huancai', P. lactiflora 'Xishifen', P. lactiflora 'Dabanhong', P. lactiflora 'Fumantang', and P. lactiflora 'Zhushapan'. Furthermore, the clustering classification results demonstrated that benzene,1,4-dimethoxy-, exhibited the highest variable importance in projection (VIP) value of 3.153, as determined by partial least squares discriminant analysis (PLS-DA).

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    Introduction

    • The herbaceous peony, a well-known traditional flower in China[1], is characterized by its large and aesthetically pleasing flowers. The herbaceous peony is a member of the family Paeoniaceae[2], displaying notable adaptability, and significant ornamental value[3]. Studies on aromatic ornamental plants involve an examination of aromatic components, and genetic mechanisms[4], including Rosa rugosa Thunb.[5], Lilium brownii var. viridulum Baker[6], and Paeonia suffruticosa Andr.[7], Pyrus communis L.[8], Dendrobium officinale [9], Nymphaea tetragona[10], Rhododendron simsii[11], Jasminum sambac[12], studies were conducted on Chrysanthemum morifolium[13], Osmanthus fragrans[14], Camellia japonica[15], Malus[16], and Iris tectorum Maxim.[17] Historically, research has focused on factors such as flower shape, color, blooming season, and resilience, with less attention given to the floral scent[18].

      The floral scent has been identified as a significant ornamental attribute of herbaceous peony[3,19], and is also a prominent feature in numerous plant species[20]. It is frequently described as the 'essence of flowers'[21] and is derived from a range of volatile compounds that are synthesized within the plant and subsequently released into the atmosphere[22]. To date, over 1,700 volatile compounds have been identified in a variety of plants, with a multitude of applications in the manufacture of perfumes, cosmetics, culinary seasonings, and pharmaceuticals[23,24]. The composition and concentration of these volatile compounds exhibit considerable variation across different species, genus, and cultivars. Nevertheless, there is a paucity of research dedicated to the analysis of fragrance constituents and their respective concentrations in herbaceous peony and tree peony[25]. Song et al.[4] identified a total of 130 volatile compounds across 30 cultivars of herbaceous peony, encompassing 72 aromatic constituents. The 24 cultivars exhibiting heightened fragrance were categorized into five distinct aroma profiles: woody scent, fruity scent, lily scent, rose scent, and an orange blossom scent. Zhao et al.[26] conducted a study in which 68 volatile compounds and 26 significant aroma constituents were identified from a sample of 87 herbaceous peony cultivars. The researchers determined that herbaceous peony contain characteristic aromatic substances, including linalool (resembling lily of the valley), geraniol (exhibiting a pleasant geranium-like scent), citronellol (evoking a fresh and light rose and leaf fragrance), and phenylethyl alcohol (noted for its distinctive rose aroma), based on the content and odor threshold of these main aroma components. In a separate study, Li et al.[27] identified 128 volatile compounds from 24 tree peony cultivars, with the predominant classes being terpenes, alcohols, and esters. The distribution pattern of these primary fragrance constituents led to the categorization of 24 tree peony cultivars into four types: grass scent (ocimene), woody scent (longifolene), lily of the valley scent (linalool), and fruity scent (2-ethyl hexanol). It has been demonstrated that the distinctive fragrances of different plant species are the result of the presence of specific volatile compounds in varying quantities and ratios. Furthermore, the quantity of fragrance emitted by flowers is contingent upon their developmental stage[28].

      Floral substances derived from plants are classified as secondary metabolites, which are released by flowering plants and predominantly comprise a range of volatile compounds characterized by relatively low molecular weights. In a comprehensive analysis of the aromatic compounds present in P. rockii and P. ostii 'Fengdan', Wu et al.[29] employed two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOF/MS). The results indicated that the aroma profile of P. rockii was primarily characterized by the presence of alcohols, alkanes, and acids, while the aroma profile of P. ostii 'Fengdan' was predominantly defined by aldehydes, alcohols, and terpenes. In a separate investigation, Li et al.[30] sought to identify and analyze the volatile compounds present in the flowers of seven pear cultivars (Anli, Bayuesu, Golden, Brown Peel, KorlaXiangli, Lyubaoshi, and Xizilü). The findings indicated that certain aldehydes constitute significant characteristics of these cultivars and are recognized as essential active odorants, which emit pronounced citrus and floral fragrances. Yang et al.[31] successfully identified and characterized 34 volatile compounds in the Dendrobium officinale flowers. Of these, 18 compounds were identified as principal odorants, including 1-octen-3-ol, hexanal, nonanal, phenylacetaldehyde, linalool, 4-oxoisophorone, theaspirane, and methyl salicylate. Furthermore, Kimani et al.[32] identified geraniol, β-caryophyllene, 2-phenylethanol, citronellol, and 1,8-cineole as the primary aromatic constituents in 24 cultivars of herbaceous peony, including P. lactiflora 'LianTai' and P. lactiflora 'Hongyan Feishuang'. Aromatic compounds are recognized as the primary chemical constituents of aromatic plants, playing a crucial role in the synthesis of secondary metabolites[33], and fragrance development. These compounds exhibit a diverse range of forms. For example, phenethyl alcohol is found in rose, mint contains menthol, and lemon includes citric acid[22,34,35].

      This study employed a combination of dynamic headspace sampling technology[36] and GC-MS to analyze the volatile components and concentrations in 100 international herbaceous peony cultivars during the half-opening stage. The objective was to elucidate the aromatic profile of the herbaceous peony. The findings of this study establish a fundamental framework for further investigation and exploitation of the fragrances of herbaceous peony flowers and provide a valuable resource for enhancing the economic value of herbaceous peony.

    Materials and methods

      Plants

    • The experimental materials used in this study were obtained from the Luoyang Academy of Agriculture and Forestry Sciences (Luoyang City, Henan Province, China) between 20 April and 8 May 2022. The majority of the materials were collected between 10 and 12 am. The subjects of the experiment were herbaceous peony plants sourced from the herbaceous peony resource garden affiliated with the Henan University of Science and Technology. As outlined in Table 1 and Fig. 1, herbaceous peony cultivars demonstrating consistent growth patterns and flowering stages were identified, and the methodology entailed the repetition of each sample on three occasions.

      Table 1.  Names and numbers of 100 herbaceous peony cultivars.

      100 herbaceous peony cultivars
      'Hangbaishao' 'Hongrongqiu' 'Biandihong' 'Zijin Daipao' 'Zixia Yingxue' 'Fenchi Dicui' 'Xishifen' 'Yinlong Hanzhu' 'Yinxian Xiuhongpao' 'Jindaiwei'
      'Luhong' 'Xueyuan Hongxing' 'Mozijin' 'Yahong' 'Wulong Tanhai' 'Hongyan Zhengshuang' 'Xingguang Chanlan' 'Yanlihong' 'Hongling Chijin' 'Fenzhuangyuan'
      'Taohua Huancai' 'Zhongshenghong' 'Ziling' 'Luxihong' 'Zifurong' 'Hongling Chijin' 'Huguang Shise' 'Hongyuqiu' 'Yanzhi Dianyu' 'Lantian Piaoxiang'
      'Zhushapan' 'Hongyun Yingri' 'Yanzi Xiangyang' 'Yanzhihong' 'Zaoyuanhong' 'ChilongCaifeng' 'Chaoshihong' 'Qingwen' 'Shaifugui' 'Ziyanshuang'
      'Gaoganfen' 'Qundiehui' 'Meirenmian' 'Meiju' 'Dafugui' 'Zhifeng Zhaoyang' 'Xueyuan Hongxing' 'Dahongpao' 'Zixiuqiu' 'Canglong'
      'Gaoganhong' 'Hongyan Feishuang' 'Dabanhong' 'Zifengyu' 'Hongpan Jinqiu' 'Hushui Dangxia' 'Yinlong Huihai' 'Baihuazi' 'Taohuafen' 'Wawamian'
      'Fenpanjinxing' 'Heixiuqiu' 'Shuanghonglou' 'Changshouhong' 'Hongyan Lushuang' 'Tuopan Jinhua' 'Hongling Chijin' 'Linglongyu' 'Jinzan Ciyu' 'Xiangyang Qihua'
      'Jinbian Hongge' 'Duoyezi' 'Fenzilou' 'Furong Jinhua' 'Fenkui' 'Guifei Chacui' 'Huolian Jingang' 'Hongguanfang' 'Fenmian Taohua' 'Taoranzui'
      'Zhaoyanghong' 'Hongfengyu' 'Fumantang' 'Shaonvfen' 'Danfeng' 'Liantaizi' 'Meiguihong' 'Fenfurong' 'Fenling Hongzhu' 'Fenqiu'
      'Fencuiqiu' 'FengChao Chuyu' 'Lanju' 'Jinsanhong' 'Zhaoyuanfen' 'Hongfeng' 'Qiaoling' 'Tuanye Jinqiu' 'Guohong' 'Tongquechun'
      The numbers 1−100 are listed from top to bottom, left to right respectively.

      Figure 1. 

      Morphological characteristics of 100 herbaceous peony cultivars at the half-opening stage. The order of the above pictures is relative to the order of cultivars in Table 1.

      Experimental equipment and materials

      Equipment

    • The Gas Chromatography-Mass Spectrometry System (GC8890-MS5977B) from Agilent Technologies, USA, and the Atmospheric Sampler QC-1S from the Beijing Institute of Labor Protection were utilized in the study.

      Chemicals

    • The reagents used included Tenax TA as the adsorbent, ethyl caprate, dichloromethane, pentane, n-hexane of chromatography grade, ethyl decanoate, ethyl acetate, and a standard solution of n-alkane mixture (ranging from C8 to C40) obtained from Sigma-Aldrich, USA.

      Collection methods of volatile components

    • The dynamic headspace adsorption technique employed in this study was a sampling bag (355 mm × 508 mm, Reynolds, USA) hermetically sealed at one end with an activated carbon filter tube. The bag was meticulously wrapped around a live peony flower to minimize contact and prevent damage to the bag. The bag's opposite end was connected to a Tenax TA adsorption tube (6 mm outer diameter, 100 mm length, filled with adsorbent) and an atmospheric sampler via tasteless silicone tubing. The flow rate of the atmospheric sampler was set at 400 mL·min−1 and the sampling duration was 3 h. Following the sampling period, the adsorption tube was sealed with cling film and aluminum foil, then placed in a self-sealing bag and stored in an ultra-low temperature cooler for transport to the laboratory. The sample was then eluted with n-hexane during sample processing, and the eluate was transferred to a brown sample bottle for subsequent analysis.

      The following conditions were observed in the gas chromatography (GC) analysis: the chromatographic column employed is a flexible quartz capillary column, with a length of 30 mm, an internal diameter of 0.25 mm, and a pore size of 0.25 μm. The flow rate of the column is set at 1.2 mL·min−1. The temperature of the column is maintained according to a specific protocol. It is initially set at 70 °C and held for 1 min, then increased to 136 °C at a rate of 6 °C·min−1, followed by further increases to 138 °C at a rate of 1 °C·min−1, then to 142 °C at a rate of 2 °C·min−1, and finally to 143 °C at a rate of 0.5 °C∙min−1. The temperature is increased by 5 °C·min−1 and subsequently to 160 °C at a rate of 2 °C·min−1, before reaching 250 °C at a rate of 10 °C·min−1. The injector temperature is set at 250 °C, with a carrier gas of high-purity helium at a flow rate of 1 mL·min−1. The injection mode is a split injection, with a split ratio of 9:1, and the injection volume is 2 μL.

      The following conditions were employed for the mass spectrometry (MS) analysis: The electron impact (EI) source is operated at 70 eV, with the interface temperature set to 250 °C and the ion source temperature maintained at 230 °C. The quadrupole temperature is controlled at 150 °C, and the scan range is from 25 to 400 amu.

      Analysis methods of volatile components

      Qualitative analysis

    • Before the analysis of the sample using gas chromatography, the 500 mg∙L−1 n-alkane mixed standard solution should be diluted with n-hexane at a ratio of 1:50, in accordance with the specified conditions for the chromatography. It is essential to record the retention time for each n-alkane and to compare the resulting Retention Index (RI) values with those documented in the literature to facilitate the identification of the compounds in question. The following formula is used to calculate the RI:

      RI=100×n+100×(tx−tn)/(tn+1−tn)

      The location of the aforementioned item is as follows: The retention index (RI) represents the retention time of the volatile substances under examination. The number of carbon atoms in the straight-chain alkane preceding the analyte is represented by n. The retention time of the analyte is represented by tx. The retention time of the straight-chain alkane before the analyte is represented by tn. The retention time of the straight-chain alkane following the analyte is represented by tn+1. The retention time of the analyte falls between the retention times of tn and tn+1. Qualitative analysis of volatile components is conducted through consultation with the NIST 17 spectral library, with cross-referencing of RI values, literature sources, and other pertinent resources, including books.

      Quantitative analysis

    • An internal standard solution, comprising 69.32 mg∙L−1 of ethyl decanoate in ethyl acetate, is employed. A volume of 0.4 μL of the internal standard solution is added to each 80 μL sample. Subsequently, quantitative calculations are performed in accordance with the following formula:

      Contentofeacharomasubstance(μg⋅g−1)=PeakareaofeacharomasubstancePeakareaoftheinternalstandard×Concentrationoftheinternalstandard(mg⋅L−1)×Volumeoftheinternalstandard(μL)Volumeofthesample(g)×f

      where, f is the correction factor of each component to the internal standard, f = 1.

      Data cleaning and analysis

    • The analysis of variance can be conducted using the statistical software package SPSS, while graph plotting can be accomplished with the Origin 2022 software. The software Metaboanalyst and the Microbiome Analysis Platform are capable of performing data normalization, partial least squares discriminant analysis (PLS-DA), and cluster analysis.

    Results

      Examination of the primary volatile compounds in various cultivars of herbaceous peony during the half opening stage

    • An analysis was conducted to determine the main volatile compounds present in 100 herbaceous peony cultivars during the half-opening stage. This was achieved through the utilization of database retrieval and manual identification methods. The results are outlined in Table 2. A total of 16 volatile components were identified and classified into six distinct groups. The data revealed that alkanes constituted six types, representing 37.5% of the total volatile components. This was followed by four types of esters at 25%, three types of alcohols at 18.75%, and one type each of terpenes, ethers, and phenols, each accounting for 6.25% of the total volatile components. The results of this analysis indicate that the predominant categories of volatile compounds found in herbaceous peony cultivars are alkanes, esters, and alcohols.

      Table 2.  The volatile components of 100 herbaceous peony cultivars.

      Compound number RT (min) CAS number Compounds Compound classification Chemical formula Retention index
      Calculated value Reference value
      1 3.273 111-84-2 Nonane Alkanes C9H20 900 900
      2 4.805 124-18-5 Decane Alkanes C10H22 1,000 1,000
      3 5.727 13877-91-3 (Z)-β-ocimene Terpenes C10H16 1,038 1,037
      4 7.063 60-12-8 Phenylethyl alcohol Alcohols C8H10O 1,115 1,109
      5 8.133 150-78-7 Benzene,1,4-dimethoxy- Ethers C8H10O2 1,165 1,168
      6 9.502 106-22-9 Citronellol Alcohols C10H20O 1,228 1,228
      7 10.084 106-25-2 Nerol Alcohols C10H18O 1,220 1,219
      8 10.187 103-45-7 Methyl cinnamate Esters C10H12O2 1,260 1,258
      9 12.976 103-26-4 2-Propenoic acid,3-phenyl-,methyl ester Esters C10H10O2 1,389 1,380
      10 14.88 131-11-3 Dimethyl phthalate Esters C10H10O4 1,456 1,466
      11 16.174 629-62-9 Pentadecane Alkanes C15H32 1,500 1,500
      12 16.664 128-37-0 Butylated hydroxytoluene Phenols C15H24O 1,513 1,513
      13 19.877 544-76-3 Hexadecane Alkanes C16H34 1,600 1,601
      14 24.137 629-78-7 Heptadecane Alkanes C17H36 1,699 1,700
      15 31.517 84-74-2 Dibutyl phthalate Esters C16H22O4 1,964 1,907
      16 33.398 646-31-1 Tetracosane Alkanes C24H50 2,400 2,400

      Comparison of volatile compounds content in different cultivars of herbaceous peony during the half opening stage

      Alkane compounds

    • As illustrated in Fig. 2, alkane compounds were undetected in 30 cultivars, including P. lactiflora 'Hushui Dangxia', P. lactiflora 'Tuopan Jinhua', P. lactiflora 'Qiaoling', P. lactiflora 'Yinlong Hanzhu', and P. lactiflora 'Yanlihong'. Among the 100 herbaceous peony cultivars, the highest concentration of alkane compounds was observed in P. lactiflora 'Heizijin' (10.66 ± 2.01 μg·g−1), with the range of alkane compounds concentration spanning from 0.00 to 10.66 μg·g−1.

      Figure 2. 

      Comparative heat map depicting the release of six types of volatile compounds from various herbaceous peony cultivars.

      Ester compounds

    • As shown in Fig. 2, ester compounds were discernible in all 44 cultivars of herbaceous peony at the half-opening stage. However, the content of ester compounds was generally not notably high in most cultivars. The highest ester compounds content was observed in P. lactiflora 'Changshouhong' (9.15 ± 0.03 μg·g−1), followed by P. lactiflora 'Zaoyuanhong' (3.55 ± 0.40 μg·g−1), P. lactiflora 'Hongyun Yingri' (3.37 ± 0.11 μg·g−1), and P. lactiflora 'Saifugui' (3.25 ± 0.67 μg·g−1). The ester compounds content among these three cultivars was found to be similar, with a range of 0.00 to 9.15 μg·g−1.

      Alcohol compounds

    • As depicted in Fig. 2, the majority of the 100 cultivars of herbaceous peony at the half-opening stage exhibited the presence of alcohol compounds. Only 23 cultivars, including P. lactiflora 'Taohua Huancai', P. lactiflora 'Zhushapan', and P. lactiflora 'Gaoganhong' exhibited no detection. The highest alcohol compounds content was observed in P. lactiflora 'Hongfeng' (22.98 ± 3.86 μg·g−1), which was significantly higher than that of other herbaceous peony cultivars. Subsequently, P. lactiflora 'Wandai Shengse' (16.23 ± 2.28 μg·g−1) exhibited the second-highest alcohol compounds content, with a range of 0.00 to 22.98 μg·g−1.

      Terpene compounds

    • As illustrated in Fig. 2, only 19 of the herbaceous peony cultivars exhibited detectable levels of terpene compounds, with significant differences in content (p < 0.05). The highest content was observed in P. lactiflora 'Hongfengyu' (8.19 ± 1.02 μg·g−1), followed by P. lactiflora 'Wandai Shengse' (4.93 ± 0.09 μg·g−1), P. lactiflora 'Jinzan Ciyu' (2.92 ± 1.75 μg·g−1), P. lactiflora 'Dabanhong' (0.07 ± 0.13 μg·g−1), P. lactiflora 'Jinbian Hongge' (0.14 ± 0.23 μg·g−1), and P. lactiflora 'Mozi Hanjin' (0.16 ± 0.28 μg·g−1), among others. The range of terpene compounds content was found to vary from 0.00 to 8.19 μg·g−1.

      Ether compounds

    • The analysis of 50 herbaceous peony cultivars revealed the presence of ether compounds in all samples, with notable variations in their content (p < 0.05). The highest content of ether compounds was observed in P. lactiflora 'Dabanhong' (22.84 ± 2.15 μg·g−1), followed by P. lactiflora 'Taohua Yingcai' (19.53 ± 2.44 μg·g−1). The lowest levels were observed in P. lactiflora 'Danfeng' (0.06 ± 0.11μg·g−1), P. lactiflora 'Ziling' (0.15 ± 0.26 μg·g−1), and P. lactiflora 'Huolian Jingang' (0.12 ± 0.21 μg·g−1). The range of ether compounds content was observed to vary from 0.00 to 22.84 μg·g−1.

      Phenol compounds

    • The analysis revealed that only five herbaceous peony cultivars exhibited discernible levels of phenol compounds, namely P. lactiflora 'Jinbian Hongge' (0.15 ± 0.05 μg·g−1), P. lactiflora 'Zhaoyanghong' (0.34 ± 0.02 μg·g−1). The remaining cultivars exhibited lower levels of phenol compounds, with the lowest concentration observed in P. lactiflora 'Hongrongqiu' (0.17 ± 0.03 μg·g−1), followed by P. lactiflora 'Xueyuan Honghua' (0.01 ± 0.02 μg·g−1), and P. lactiflora 'Ziling' (0.27 ± 0.05 μg·g−1). The five cultivars exhibited notably lower levels of phenol compounds, with values consistently below 1 μg·g−1. The remaining cultivars were found to be devoid of phenol compounds.

      An analysis was conducted on the total release of aroma components in different herbaceous peony cultivars

    • The analysis of the 16 volatile compounds detected revealed that, aside from alkanes such as nonane, the remaining 10 compounds from five classes all exhibited characteristic aromas, as detailed in Table 3. These aromatic compounds were present in the majority of samples, with concentrations exceeding 0.01 μg·g−1. Of particular note is the detection of benzene,1,4-dimethoxy-, in the majority of samples, with relatively high concentrations observed (Fig. 3).

      Table 3.  Characteristics of aroma compounds.

      No. Compound name Odor characteristics
      1 (Z)-β-ocimene The scent of grass and flowers is accompanied by the aroma of orange blossom oil[37]
      2 Phenylethyl alcohol Sweet rose-like fragrance[38]
      3 Benzene,1,4-dimethoxy- The fragrance of cloves[39]
      4 Citronellol Has a sweet rose aroma[40]
      5 Nerol There is a sweet rose fragrance[41]
      6 Acetic acid, 2-phenylethyl ester There is a reminiscent of honey-like floral fragrance[42]
      7 Methyl cinnamate Sweet smelling fragrance[43]
      8 Dimethyl phthalate The substance emits a delicate fragrance[44]
      9 Butylated hydroxytoluene The presence of a carbonic acid taste can
      influence the aroma of wine[45]
      10 Dibutyl phthalate The substance emits a delicate fragrance[46]

      Figure 3. 

      Content of characteristic aroma compounds in herbaceous peony cultivars.

      Cluster analysis of aromatic components in various herbaceous peony cultivars

    • A data matrix of dimensions 100 × 10 was constructed, representing the content of 10 aromatic compounds in 100 herbaceous peony cultivars as variables. A cluster heatmap was generated using the microbiome analysis platform, as illustrated in Fig. 4. In light of the clustering results and a comprehensive consideration of the major aromatic components, the 100 herbaceous peony cultivars are ultimately classified into two groups (Table 4). The first group of herbaceous peony cultivars is distinguished by a marked prevalence of benzene,1,4-dimethoxy-, with markedly elevated levels in comparison to other cultivars. This gives rise to a pronounced clove scent, indicative of a clove floral type. This initial classification is based on the presence of specific compounds and is therefore applicable to only five cultivars. The cultivars in question are P. lactiflora 'Taohua Huancai', P. lactiflora 'Xishifen', P. lactiflora 'Dabanhong', P. lactiflora 'Fumantang', and P. lactiflora 'Zhushapan'. The second group generally exhibits lower levels of aromatic compounds, resulting in milder scents that may be characterized as a light floral type. The second group comprises 95 cultivars, including representative cultivars such as P. lactiflora 'Meiju', P. lactiflora 'Shaonvfen', P. lactiflora 'Fenmian Taohua', P. lactiflora 'Fenling Hongzhu', and P. lactiflora 'Guohuo', among others.

      Figure 4. 

      Heat map showing the clustering analysis of 100 herbaceous peony cultivars. A-Benzene,1,4-dimethoxy-, B-Citronellol, C-Nerol, D-Acetic acid, 2-phenylethyl ester, E-Methyl cinnamate, F-Dimethyl phthalate, G-(Z)-β-ocimene, H-Phenylethyl alcohol, I-Butylated hydroxytoluene, J-Dibutyl phthalate. The numbers 1−100 correspond to the cultivar names listed in Table 1.

      Table 4.  Cluster analysis of characteristic aroma components in different herbaceous peony cultivars.

      Groups Herbaceous peony cultivars
      1 'Taohua Huancai', 'Xishifen', 'Dabanhong', 'Fumantang', and 'Zhushapan'
      2 'Liantaizi', 'Hushui Dangxia', 'Shaifugui', 'Hongfeng', 'Wandai Shengse', 'Zhaoyuanfen', 'Wawamian', 'Lanju', 'Shuanghonglou', 'Fenling Hongzhu', 'Guohuo', 'Fenmian Taohua', 'Yinlong Tanhai', 'Chaoshihong', 'Shaonvfen', 'Meiju', 'Huolian Jingang', 'Meiguihong', 'Chilong Huancai', 'Yinlong Hanzhu', 'Yanlihong', 'Zhaoyanghong', 'Yinxian Xiuhongpao', 'Fenchi Dicui', 'Xueyuan Hongxing', 'Fenfurong', 'Linglongyu', 'Xiangyang Qihua', 'Hongrongqiu', 'Huguang Shise', 'Yanzhihong', 'Duoyezi', 'Mozijin', 'Guifei Chacui', 'Ziling', 'Zixia Yingxue', 'Zixiuqiu', 'Jinzan Ciyu', 'Meirenmian', 'Zifengyu', 'Jinshanhong', 'Hongyan Lushuang', 'Hongguanfang', 'Jindaiwei', 'Canglong', 'Tuopan Jinhua', 'Huolian Chijin', 'Fengchao Chuyu', 'Hongyuqiu', 'Xueyuan Hongxing', 'Qiaoling', 'Dahongpao', 'Qundiehui', 'Tuanye Jinqiu', 'Dafugui', 'Taoranzui', 'Yanzhi Dianyu', 'Tongquechun', 'Ziyanshuang', 'Gaoganfen', 'Fenpan Jinxing', 'Fenkui', 'Lantian Piaoxiang', 'Zifeng Zhaoyang', 'Xingguang Canlan', 'Hongyan Feishuang', 'Biahuazi', 'Taohuafen', 'Danfeng', 'Hongfengyu', 'Fenzilou', 'Yanzi Xiangyang', 'Zaoyuanhong', 'Luhong', 'Yahong', 'Luxihong', 'Furong Jinhua', 'Jinbian Hongge', 'Wulong Tanhai', 'Zhongshenghua', 'Zifurong', 'Hongyan Zhengshuang', 'Gaoganhong', 'Heixiuqiu', 'Hongling Chijin', 'Hongyun Yingri', 'Changshouhong', 'Fencuiqiu', 'Qingwen', 'Hongpan Jinqiu', 'Zijin Daipao', 'Biandihong', 'Fenqiu', 'Hangbaishao' and 'Fenzhuangyuan'

      Partial Least Squares Discriminant Analysis (PLS-DA)

    • Following the clustering of 100 cultivars into two groups, a partial least squares discriminant analysis (PLS-DA) was conducted on the content of 10 aroma compounds in the 100 cultivars using Metaboanalyst software. The results of the analysis are presented in Fig. 5. The PLS model for aroma compounds demonstrated satisfactory reliability, as evidenced by R2 and Q2 values of 0.702 and 0.598, respectively. Moreover, the PLS-DA results demonstrated variations in the profile of aroma compounds between the two groups of cultivars (Fig. 5a). The application of a VIP criterion greater than 1 identified a differentiating component (Fig. 5b). The VIP values in the PLS-DA model provided further insight into the contribution of each component to the model, with components having a value of VIP > 1 being considered significant. For instance, benzene,1,4-dimethoxy-, exhibited a VIP value of 3.153 and was identified as a principal component accountable for the discrepancies among herbaceous peony cultivars (Fig. 5b), corroborating the findings of the clustering analysis. It can therefore be posited that benzene,1,4-dimethoxy- is a characteristic aroma component of these herbaceous peony cultivars.

      Figure 5. 

      PLS-DA scores of 100 herbaceous peony cultivars under two cluster groups.

      Variables A and B represent the first and second categories, respectively. The specific variables include A-Benzene,1,4-dimethoxy-, B-Citronellol, C-Nerol, D-Acetic acid, 2-phenylethyl ester, E-Methyl cinnamate, F-Dimethyl phthalate, G-(Z)-β-ocimene, H-Phenylethyl alcohol, I-Butylated hydroxytoluene, J-Dibutyl phthalate.

    Discussion

    • The present study comprises a comprehensive identification and analysis of the volatile constituents present in 100 herbaceous peony cultivars during the half-opening stage. The findings indicated that alkanes, alcohols, and ethers were the most prevalent volatile compounds, with benzene,1,4-dimethoxy- was identified as the distinctive aromatic components.

      One such molecule is benzene,1,4-dimethoxy-, a methoxylated aromatic volatile compound that is known to elicit physiological and behavioral responses in a diverse range of insect pollinators. It serves as a principal floral volatile in a number of plant species belonging to diverse genera, including Salix, Lithophragma, Nelumbo, Catasetum, Allium, and Fragaria[47]. Wang et al.[40] identified the common floral component, benzene,1,4-dimethoxy-, in all eight herbaceous peony cultivars. Furthermore, Kimani et al.[32] identified 95 volatile organic compounds in 24 herbaceous peony cultivars, including benzene,1,4-dimethoxy-, which is a phenolic methyl ether containing a benzene skeleton but not derived from aromatic amino acids. Rather, it is a member of a particular chemical class that is responsible for the olfactory characteristics of specific plant varieties. The types and contents of volatile components of herbaceous peonies may be associated with the sampling method, sampling location and time. Additionally, the types and contents of volatile compounds in plants may be influenced by different planting environmental conditions[48].

      In recent years, there has been a growing emphasis on the natural floral volatiles present in herbaceous peonies, with the fragrance components demonstrating a diverse range of applications in the fields of healthcare, perfumes, and cosmetics[49]. Floral scent represents a significant component of plant volatiles, which are primarily composed of terpenes, aromatic hydrocarbons, fatty acids, and their derivatives, as well as sulfur and nitrogen-containing compounds[27,50]. These compounds are taxonomically categorized into three primary classes, contingent upon their biogenic origins, namely fatty acid derivatives, phenylpropanoids/benzenoids, and terpenoids[51]. In the present study, the volatile components of the 100 cultivars of herbaceous peony were predominantly identified as alkanes, esters, and alcohols. The most abundant type of compound was identified as alkanes. The available evidence suggests that straight-chain alkanes represent the primary constituents of plant leaf wax[52]. These waxes are not exclusive to leaves but may also be found on other plant organs, including flowers and fruit surfaces[53]. This indicates that wax layers may cover the surfaces of the majority of herbaceous peony cultivars. Alcohols play a significant role in the fragrance industry, serving as essential raw materials for synthetic fragrances and as an indispensable component in perfumery[54]. The presence of abundant ether compounds results in the production of pleasant floral and fruity aromas, while simultaneously enhancing the richness, typicality, and complexity of plant fragrances[55].

      However, due to the constraints of the existing literature, some volatile components, such as specific alkanes, have not yet been conclusively identified as fragrance components. Further research is required to ascertain whether these components contribute to the fragrance of herbaceous species. Alkane compounds have relatively high thresholds[56] and make minimal contributions to the overall scent[28]. Accordingly, the analysis of fragrance compounds excludes the contributions made by nonane, decane, pentadecane, hexadecane, heptadecane, and tetracosane.

    Conclusions

    • This study employed dynamic headspace bag adsorption of live plant materials and gas chromatography-mass spectrometry (GC-MS) analysis techniques to identify a total of 16 volatile components in 100 herbaceous peony cultivars at the half-opening stage[57]. The components were primarily categorized into six major groups: alkanes, esters, alcohols, terpenes, ethers, and phenols. The predominant volatile compounds were alkanes, alcohols, and ethers, while benzene,1,4-dimethoxy- was identified as the main aromatic component. Significant variations in the total content of the main aromatic components were observed among the different herbaceous peony cultivars at the half-opening stage. In particular, P. lactiflora 'Taohua Huancai', P. lactiflora 'Xishifen', P. lactiflora 'Dabanhong', P. lactiflora 'Fumantang', and P. lactiflora 'Zhushapan' exhibited the highest content of aromatic components, resulting in a more intense floral fragrance. The intensity and characteristics of the aroma exhibited notable variation among different herbaceous peony cultivars, attributable to differences in the quantity and composition of the aromatic components. This is a crucial indicator for evaluating the quality of herbaceous peony. This study provides a theoretical foundation for understanding the formation and regulation mechanisms of herbaceous peony aroma characteristics, while also offering technical support for accelerating industrial development and utilization of herbaceous peony aromas.

      Acknowledgments

      • This research was funded by the Science and Technology Innovation Talents in Universities of Henan Province (Grant No. 22HASTIT036) and the Project of Henan Province Traditional Chinese Medicine Industry Technology System (Grant No. 2024-24).

      Author contributions

      • The authors confirm contribution to the paper as follows: study conceptualization, reviewing, editing and funding acquisition: Guo L; material preparation: Wang A, Luo Y, Niu T, Zhao X, Gao K; data curation: Wang A, Luo Y, Niu T, Wang S; draft manuscript preparation: Wang A, Luo Y; manuscript reviewing and editing: Hou 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.

      Conflict of interest

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

      • # Authors contributed equally: Aixin Wang, Yasang Luo

      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (5)  Table (4) References (57)
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    Cite this article
    Wang A, Luo Y, Niu T, Gao K, Wang S, et al. 2024. Identification and content analysis of volatile components in 100 cultivars of Chinese herbaceous peony. Ornamental Plant Research 4: e032 doi: 10.48130/opr-0024-0029
    Wang A, Luo Y, Niu T, Gao K, Wang S, et al. 2024. Identification and content analysis of volatile components in 100 cultivars of Chinese herbaceous peony. Ornamental Plant Research 4: e032 doi: 10.48130/opr-0024-0029
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  • Introduction
  • Materials and methods

  • Plants

  • Experimental equipment and materials

  • Equipment
  • Chemicals

  • Collection methods of volatile components

  • Analysis methods of volatile components

  • Qualitative analysis
  • Quantitative analysis

  • Data cleaning and analysis

  • Results

  • Examination of the primary volatile compounds in various cultivars of herbaceous peony during the half opening stage

  • Comparison of volatile compounds content in different cultivars of herbaceous peony during the half opening stage

  • Alkane compounds
  • Ester compounds
  • Alcohol compounds
  • Terpene compounds
  • Ether compounds
  • Phenol compounds

  • An analysis was conducted on the total release of aroma components in different herbaceous peony cultivars

  • Cluster analysis of aromatic components in various herbaceous peony cultivars

  • Partial Least Squares Discriminant Analysis (PLS-DA)

  • Discussion
  • Conclusions
  • Acknowledgments
  • Author contributions
  • Data availability
  • Conflict of interest
  • Rights and permissions
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