Skip to main content

Comparison of chrysanthemum flowers grown under hydroponic and soil-based systems: yield and transcriptome analysis

Abstract

Background

Flowers of Chrysanthemum × morifolium Ramat. are used as tea in traditional Chinese cuisine. However, with increasing population and urbanization, water and land availability have become limiting for chrysanthemum tea production. Hydroponic culture enables effective, rapid nutrient exchange, while requiring no soil and less water than soil cultivation. Hydroponic culture can reduce pesticide residues in food and improve the quantity or size of fruits, flowers, and leaves, and the levels of active compounds important for nutrition and health. To date, studies to improve the yield and active compounds of chrysanthemum have focused on soil culture. Moreover, the molecular effects of hydroponic and soil culture on chrysanthemum tea development remain understudied.

Results

Here, we studied the effects of soil and hydroponic culture on yield and total flavonoid and chlorogenic acid contents in chrysanthemum flowers (C. morifolium ‘wuyuanhuang’). Yield and the total flavonoids and chlorogenic acid contents of chrysanthemum flowers were higher in the hydroponic culture system than in the soil system. Transcriptome profiling using RNA-seq revealed 3858 differentially expressed genes (DEGs) between chrysanthemum flowers grown in soil and hydroponic conditions. Gene Ontology (GO) enrichment annotation revealed that these differentially transcribed genes are mainly involved in “cytoplasmic part”, “biosynthetic process”, “organic substance biosynthetic process”, “cell wall organization or biogenesis” and other processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed enrichment in “metabolic pathways”, “biosynthesis of secondary metabolites”, “ribosome”, “carbon metabolism”, “plant hormone signal transduction” and other metabolic processes. In functional annotations, pathways related to yield and formation of the main active compounds included phytohormone signaling, secondary metabolism, and cell wall metabolism. Enrichment analysis of transcription factors also showed that under the hydroponic system, bHLH, MYB, NAC, and ERF protein families were involved in metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction.

Conclusions

Hydroponic culture is a simple and effective way to cultivate chrysanthemum for tea production. A transcriptome analysis of chrysanthemum flowers grown in soil and hydroponic conditions. The large number of DEGs identified confirmed the difference of the regulatory machinery under two culture system.

Peer Review reports

Background

Chrysanthemum (Chrysanthemum × morifolium Ramat.) originated in China and is a popular ornamental plant with a long history of cultivation [1]. It has been used for centuries as an important traditional Chinese tea, with reported anti-inflammation, antioxidant, and antiallergic properties [2, 3]. Traditional Chinese chrysanthemum tea is prepared by boiling chrysanthemum flowers in water to extract the beneficial compounds. Several studies have demonstrated that this chrysanthemum extract has strong antioxidant properties, and inhibitory effects against bacteria and viruses [3,4,5,6,7]. Flavonoids and chlorogenic acids have potent antioxidant activity and are among the biologically active components of chrysanthemum tea [2,3,4,5,6,7,8].

With the progress of industrialization and urbanization, land and water resources are increasingly scarce. Favorable environmental condition with quality sources such as soil and water are mainly supply the production of food crops. So being important economic crops, tea and medicinal chrysanthemum are in a disadvantageous state in terms of planting area and soil environment. Moreover, continuous cultivation of crops such as chrysanthemum in large areas can lead to soil-borne diseases, nutrient deficiency, and eventually declining yield and quality [9, 10]. Many studies have focused on different approaches to improve chrysanthemum yield and quality, including screening different varieties and developing new germplasm [2, 5, 11, 12], adjusting fertilizer ratios [13,14,15], testing the effect of abiotic stress [16], and examining the effect of different extraction methods on the activity of active compounds [3, 6]. However, these studies did not address issues associated with pressures on land use, or solve fundamental problems of soil and other aspects of the growth environment.

Hydroponic culture does not use soil, and so is not affected by soil-borne diseases or soil nutrient deficiencies. It also requires less water, fertilizer, and pesticides compared with cultivation in soil [17]. Changing the hydroponic medium enables adjustment of plant nutrients at any time. Hydroponic culture has been used for many cereal crops [18,19,20,21,22], fruits [22], vegetables [23,24,25,26,27,28,29], and flowers [30]. In some cases, hydroponic cultivation improves yield and quality [22,23,24,25,26,27,28,29,30]. Studies have explored the application of hydroponic technology to chrysanthemum, mainly to determine a suitable growth medium [30]. As a health food and tea, chrysanthemum is important for human disease resistance [3, 7, 31, 32]; therefore, the production of more chrysanthemum flowers with higher quality and less pesticide residues under controlled environmental conditions has become a top priority for chrysanthemum research. However, few studies have sought to understand the physiological and gene regulatory network differences of chrysanthemum grown in two culture systems.

Flower bud differentiation or development are important tissue to chrysanthemum for tea. Flowering is a complex process controlled by gene expression and phytohormones [33,34,35,36,37,38,39,40]. In chrysanthemum, important transcription factors (TFs) involved in flower development have been isolated and analyzed, including those encoded by the homologs of the Arabidopsis genes APETALA1, SEPALLATA3, FRUITFULL, LEAFY, APETALA2,TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR20, and CYCLOIDEA2c, as well as many genes encoding MADS-box and WUSCHEL (WUS)-like proteins [33,34,35,36,37,38,39,40]. In the current study, many members of different TF families, such as WRKY, MYB, MYC, NAC, C3H, Trihelix, C2H2, ERF and CRT TFs were upregulated during flower bud differentiation or development of chrysanthemum flower [35,36,37,38,39,40]. In sweet osmanthus (Osmanthus fragras Lour), low temperature increased the expression of genes related to cell expansion (encoding Expansin,

Xyloglucan endotransglycosylase/hydrolase, Xylosidase, Polygalacturonase) and phytohormones (Auxin, Gibberellic acid, Ethylene, and Brassinosteroids) to affect flowering time [41]. Transcription of expansin genes increased during flower opening of wintersweet (Chimonanthus paraecox) [42].

For foods with health-promoting properties, the level of bioactive compounds is an important component of quality. The flavonoid and chlorogenic acid biosynthetic pathways have largely been characterized [43]. Functional structural genes and regulatory gene pathways are conserved in plants [44]. Many functional genes (encoding chalcone synthase, chalcone isomerase, favone synthase, favanone 3-hydroxylase, favonoid 3′-hydroxylase, favonoid 3′,5′-hydroxylase, favonol synthase, dihydrofavonol 4-reductase, anthocyanidin synthase and UDP-glucose-favonoid 3-O-glucosyltransferase) in the flavonoid biosynthetic pathway have been identified in chrysanthemum. Additional, in C. morifolium ‘Chuju’, MYB–bHLH–WD40 TF complexes regulate flavonoid biosynthesis during the beginning of flower bud formation [44].

In recent years, RNA-seq has been used for transcriptomic analysis of flower development [35,36,37,38,39, 43], secondary metabolite synthesis [44,45,46], and stress tolerance [47,48,49] in chrysanthemum and its wild relatives [50, 51]. Such analysis has helped to reveal the molecular mechanisms of various processes and discover many candidate genes (homologs of Early Flowering, Late Elongated Hypocotyl, Pseudo-Tesponse Regulators, Circadian Clock Associated, Chalcone Synthase, Chalcone Isomerase, Favone Synthase, Favanone 3-hydroxylase, Favonoid 3′-hydroxylase, Favonoid 3′,5′-hydroxylase, Favonol Synthase, Dihydrofavonol 4-reductase, Anthocyanidin Synthase and UDP-glucose-favonoid 3-O-glucosyltransferase,et al.,) for various type of conditions. However, chrysanthemum has many hybrid polyploid varieties that lack high-quality reference genome information. Fortunately, whole-genome fine mapping of C. indicum ‘Nankingense’ has been established using next-generation sequencing [52]. C. indicum is a diploid plant and a wild ancestor of Chrysanthemum × morifolium (Ramat.) [1]. Whole-genome fine mapping of this variety provides a reliable reference genome for transcriptome analysis of chrysanthemum.

Hydroponic culture can improve the quantity or size of fruits, flowers and leaves, as well as increase production of the nutritionally and medicinally important active compounds in many crops, fruits, vegetables, and flowers [22,23,24,25,26,27,28,29,30,31]. Therefore, the transcription of genes related to flower development and active compound biosynthesis might be upregulated in response to hydroponic culture. To test this, in the current study, we used RNA-seq to identify differentially expressed genes (DEGs) involved in metabolic pathways in two culture systems. Identification of DEGs in chrysanthemum may provide new genetic resources for this species. This study will help reveal the molecular mechanisms underlying chrysanthemum yield and qualities associated with hydroponic culture.

Results

Comparison of hydroponic and soil-grown chrysanthemum

Hydroponically cultivated chrysanthemum plants grew normally (Fig. 1A, B, C). When harvested for comparison (Fig. 1D, E), the average number and dry weight of hydroponically cultivated chrysanthemum flowers were greater than field-cultivated ones (41.33 ± 11.64 flowers per plant, and 20.25 ± 5.70 g dry weight per flower for hydroponic cultivation versus 25.67 ± 3.43 flowers per plant, and 12.58 ± 1.68 g dry weight for soil cultivated flowers, meaning increased production by up to 37.89% with hydroponic cultivation).

Fig. 1
figure1

Phenotype, number and dry weight of chrysanthemum under soil system and hydroponic system. A Phenotype of chrysanthemum at seeding stage under Soil cultivation; B Phenotype of chrysanthemum at seeding stage under Hydroponic cultivation; Phenotype of chrysanthemum at blooming stage under Soil system(left) and hydroponic system(right); D Comparative number of flowers from one plant of chrysanthemum grown under soil system and hydroponic system.*P < 0.05, **P < 0.01; E Comparative dry weight of flowers from one plant of chrysanthemum grown under soil system and hydroponic system. *P < 0.05, **P < 0.01

Chrysanthemum tea extracts from flowers grown under soil or hydroponic systems showed differences in their biologically active components. Hydroponically cultivated chrysanthemum flowers water extracts achieved 37.8% ± 4.47, which was higher than soil-cultivated chrysanthemum flowers (33.4% ± 10.45) (Fig. 2C). Total flavonoids and chlorogenic acid content of chrysanthemum flowers grown in soil and hydroponic systems are shown in Fig. 2A, B. Flowers grown hydroponically had higher flavonoid and chlorogenic acid contents (43.22 ± 2.05 mg/g and 2.330 ± 0.40 mg/g dry weight [DW], respectively) than flowers grown in soil (36.75 ± 3.50 mg/g and 1.715 ± 0.32 mg/g DW, respectively).

Fig. 2
figure2

Comparative characterization of chrysanthemum grown by hydroponic system and soil system for flower quality. A Difference of total flavonoids content in chrysanthemum flower under soil and hydroponic system. *P < 0.05, **P < 0.01; B Difference of total chlorogenic acid content in chrysanthemum flower under soil and hydroponic system. *P < 0.05, **P < 0.01; C Difference of total water extract content in chrysanthemum flower under soil and hydroponic system. *P < 0.05, **P < 0.01

RNA-seq analysis of chrysanthemum flowers grown in hydroponic and soil systems

To better understand how different culture systems affect molecular mechanisms in chrysanthemum flower, total RNA from chrysanthemum flowers grown in soil and hydroponic systems was sequenced using an Illumina system. Transcriptomic data of six samples were obtained, with three biological replications for each condition. RNA-seq analysis provided 41.9–47.5 million raw reads per biological replicate, with an average read length of 100 bp (Table 1). After filtering out adapters, low-quality reads, virus reads and unblasted reads, approximately 133.1 million clean reads were obtained across the six transcriptome libraries. Q20 percentages of each sample (sequencing error rates lower than 1%) were higher than 98.17%. Meanwhile, 77.69–87.09% of clean reads could be aligned with the C. nankingense reference genome [53], 71.89–80.52% could be accurately mapped to a specific location within the reference genome sequence, and 5.78–6.57% mapped to multiple locations. The gene expression level could be quantified by normalized fragments per kilobase of transcript per million mapped reads (FPKM) [53]. FPKM data were tested to evaluate correlations among biological replicates; all the Pearson correlation coefficients among biological replicates were higher than 0.95 (Figure S1). In addition, principal component analysis (PCA) was performed to visualize the variation among samples utilizing DESeq2 [54], and the biological replications for each sample were clustered together (Figure S2). Overall, the transcriptomic data in this work were viable on the basis of statistical analysis from chrysanthemum flower RNA sequencing.

Table 1  Summary of the sequence data from RNA sequencing

Identification and analysis of DEGs

Chrysanthemum flowers grown under soil and hydroponic systems were compared to identify DEGs (q-value≤0.05 and |log2FC| ≥ 1) by DEGseq2 software. A total of 3858 genes were differentially expressed in chrysanthemum flowers grown under soil and hydroponic systems, with 1556 upregulated (Table S5) and 2306 downregulated in plants grown soil compared with hydroponically-grown plants (Fig. 3A, Table S6).

Fig. 3
figure3

The expression profile of DEGs in flowers of chrysanthemum under two culture systems

Based on hierarchical clustering analysis of gene expression, 3858 DEGs could be roughly grouped into four classes (Fig. 4). A total 104 DEGs were specifically expressed in chrysanthemum flowers grown in the hydroponic system, including many genes involved in “carbohydrate metabolic” (GO:0005975), “transcription” (GO:0006351), “fatty acid and unsaturated fatty acid biosynthetic process” (GO:0006633, GO:0006636), “auxin-activated signaling pathway” (GO:0009734), and “phenylpropanoid metabolic process” (GO:0009698). These genes may have important functions in chrysanthemum flower development. Conversely, ten DEGs were specifically expressed in chrysanthemum flowers grown in the soil system, including genes involved in “transcription” (GO:0006351), and “protein phosphorylation” (GO:0006468). More unigenes were downregulated than upregulated in plants grown soil compared with hydroponically-grown plants.

Fig. 4
figure4

Hierarchical cluster analysis of 3858 DEGs based on the log (FC) of gene expression. The color gradient from low (blue) to high (red) represents relative levels of gene expression. The numbers in the scale bar stand for the score of gene expression

GO and KEGG enrichment analysis of DEGs

To determine the fundamental functions of the obtained DEGs, GO analysis was performed using Cluster Profile R packages [55]. A total of 3858 DEGs were enriched in 266 GO terms, and could be classified into three categories: biological process, molecular function, and cellular component. In particular, there were 62 GO terms in which DEGs were enriched by a factor of more than 100 (Table S1). GO annotation revealed 62 GO terms for 29 annotations in cellular components, 27 annotations in biological processes, and 6 annotations in molecular functions. “Cytoplasm” (GO:0005737), “cytoplasmic part” (GO:0044444), “membrane” (GO:0016020), “cell periphery” (GO:0071944), and “cell wall” (GO:0005618) were significantly enriched in the cellular components category. In the biological processes category, “biosynthetic process” (GO:0009058), “organic substance biosynthetic process” (GO:1901576), “cellular biosynthetic process” (GO:0044249), and “cell wall organization or biogenesis” (GO:0071554) were enriched. In the molecular functions category, major enriched functions were “transmembrane transporter activity” (GO:0022857), “structural molecule activity” (GO:0005198), “transferase activity, transferring glycosyl groups” (GO:0016757), and “structural constituent of ribosome” (GO:0003735). Furthermore, the enriched GO terms from flowers grown under two culture systems were compared on the basis of their biological processes (Fig. 5).

Fig. 5
figure5

Categories and distribution of GO terms in the flowers under soil and hydroponic system

To identify the metabolic pathways of genes differentially expressed in the hydroponic system, we first searched the DEGs against KEGG using KofamKOALA. A total of 3858 DEGs were anchored in KEGG Orthology (KO) terms, wherein 873 DEGs were prominently enriched in 25 pathways (Fig. 6, Table S2). It is noteworthy that 246 and 160 DEGs were found enriched in two pathways, “metabolic pathways” (ko01100) and “biosynthesis of secondary metabolites” (ko02220). Furthermore, four pathways, “biosynthesis of secondary metabolites” (ko02220), “starch and sucrose metabolism” (ko00500), “plant hormone signal transduction” (ko04075) and “fatty acid degradation” (ko00071) include up- and downregulated DEGs.

Fig. 6
figure6

KEGG pathways prominently enriched DEGs in the flowers under soil and hydroponic system

Identification of DEGs involved in secondary metabolism

Chrysanthemum contains many bioactive ingredients: flavonoids, triterpenes, sterols, volatile oils and fatty acids, which are beneficial to humans. We focused on genes involved in “flavonoid biosynthesis” and “fatty acid and unsaturated fatty acid biosynthesis”. KEGG annotation results revealed many biosynthesis processes including “flavonoid biosynthesis”, “phenylpropanoid biosynthesis” and “fatty acid and unsaturated fatty acid biosynthesis” (Table S2), which were significantly enriched in flowers grown under the hydroponic system. Among key genes in the flavonoid pathway, shikimate O-hydroxycinnamoyltransferase, caffeoyl-CoA O-methyltransferase, chalcone-flavonone isomerase, and dihydroflavonol 4-reductase were the most upregulated, while caffeoyl-CoA O-methyltransferase was downregulated in flowers grown under hydroponic system conditions (Table S3, Fig. 7A). Phenylpropanoid synthesis is important since it is the initial substrate of many secondary metabolites. Phenylpropanoid biosynthesis was also enriched with many DEGs, including 24 up- and 11 downregulated (Table S4 Fig. 7B). Thirty-seven genes were identified in the fatty acid and unsaturated fatty acid biosynthesis pathway, of which 30 were significantly upregulated and 7 were significantly downregulated (Table S5, Fig. 7C).

Fig. 7
figure7

Heat maps of gene expression patterns involved in biological pathways obtained from KEGG. Heat maps were drawn according to log2FPKM in flowers under soil and hydroponic system. The rows and columns in the heat maps represent samples and genes id, respectively. Red and blue represent the highest and lowest level of expression. A Flavonoid biosynthesis; B phenylpropanoid biosynthesis; C fatty acid biosynthesis

Identification of DEGs involved in cell wall metabolism

Flower growth depends on cell wall loosening and cellulose biosynthesis, soluble carbohydrate allocation and cytoskeleton rearrangement [56, 57]. Therefore, we screened for DEGs that might be involved in cell wall metabolism under different culture systems. In our study, seven expansin genes, six xyloglucan endotransglucosylase/hydrolase genes, five polygalacturonase genes and seven pectate lyase genes were upregulated under hydroponic conditions, suggesting that these genes are important for regulating chrysanthemum flower growth. Four xylosidases were down-regulated undrer soil conditions and 6 of them up-regulated. Eighteen pectinesterase were up-regulated under hydroponic conditions except CHR00007443, CHR00007160, CHR00007163 and CHR00092708 (Table S6, Fig. 8).

Fig. 8
figure8

Heat maps of cell wall metabolism related genes in flowers. Heat maps were drawn according to log2FPKM in flowers under soil and hydroponic system. The rows and columns in the heat maps represent samples and genes id, respectively. Red and blue represent the highest and lowest level of expression. A EXP: expansin gene, B PE: pectinesteraseg gene; C PL: pectate lyase gene; D PG: polygalacturonase gene; E XTH: xyloglucan endotransglucosylase/hydrolase gene; F XYL: xylosidase gene

Transcription factor upregulation occurred more in hydroponically grown flowers

We further analyzed TFs to better understand the regulatory network of chrysanthemum flowers grown under both culture systems. Transcriptome sequencing results showed that 2298 and 1580 TFs were up- and downregulated, respectively, in flowers under hydroponic conditions, and these were classified into 52 transcription factor families. Expression of many TFs changed dramatically under different growth systems, so we improved the screening criteria. TFs with a |log2Ratio| ≥ 2 in any comparison were analyzed further (Table S7). The highest number of TFs belonged to the bHLH (83, 10.64%), MYB (82, 10.51%), NAC (67, 8.59%), and ERF (52, 6.67%) families. Figure 9 shows bar graphs of the top 12 transcription factor families and a comparison of up- and downregulated TFs in flowers grown under soil and hydroponic conditions. A small proportion of TF genes were downregulated under hydroponic conditions. These important TF genes were substantially differentially expressed between the two growth conditions, and may be crucial for the differences in flower development.

Fig. 9
figure9

Determination of the top 12 transcription factor families in flowers under soil and hydroponic system. Blue represents the up-regulated TFs, yellow represents the down-regulated TFs, and the vertical axis shows the number of TFs

Validation of RNA-seq analysis by qRT-PCR

To determine the reliability of DEGs obtained from RNA-seq analysis of chrysanthemum flowers, 11 DEGs were selected for qRT-PCR analysis that participated in different biological pathways including “flavonoid biosynthesis”, “plant hormone signal transduction”, “photosynthesis” (Table S8), wherein GAPDH was used as the reference gene for normalization. As a result (Fig. 10), high correlation (R2 = 0.557) between qRT-PCR and RNA-seq was observed. This result unambiguously confirmed the reliability of the DEGs obtained from RNA-seq analysis in this study.

Fig. 10
figure10

Verification of RNA-Seq results by qRT-PCR. A Relative expression ratio of each DEG is presented in -log2(Fold Change). B Correlation between RNA-Seq and qRT-PCR

Discussion

Many plants can grow in hydroponic systems [17,18,19,20,21,22,23,24,25,26,27,28,29,30]. For crops, the quality of produce, taste and the nutritive value of the end products is generally higher in hydroponic systems than in natural soil cultivation. Hydroponic culture can avoid problems such as the reduction of high-quality land area, shortage of fresh water resources, and reduction of yield caused by soil-borne diseases. There has been much research into how hydroponic culture systems affect plant growth and development, but their effect on growth, development, and regulation of molecular network mechanisms remains unclear for chrysanthemum flower.

In the present study, yield and quality of chrysanthemum flowers grown under a hydroponic culture system were higher than those grown in a soil system. RNA-seq technology was used to explore the effects of hydroponic culture on chrysanthemum flower. Transcriptome sequencing was used to analyze the flower, leaf and root under light, salinity and other conditions of chrysanthemum, and many key genes were also found [35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51]. Through KEGG and GO enrichment analysis, key genes and biological pathways were screened to comprehensively analyze the effects of hydroponic culture on gene expression and regulatory networks. The identification of DEGs in this species may provide new genetic resources for improving yield and quality of chrysanthemum.

Growth and development regulation of flowers

In this study, we analyzed the number and dry weight of chrysanthemum flowers grown in different culture systems. Our results indicated that the hydroponic system was more efficient for growing chrysanthemums than soil culture. Flower growth is accompanied by cell division and cell expansion in petal cells [56, 57]. Cell wall synthesis, modification or hydrolysis; cell expansion and loosening; and rearrangement of cell wall fibers in growing tissues probably participate in the process of flower growth [58,59,60]. Similarly, during growth of chrysanthemum flowers in a hydroponic system, seven expansin genes, six xyloglucan endotransglucosylase/hydrolase genes, five polygalacturonase genes, and seven pectate lyase genes were upregulated, and partial xylosidase, cellulose synthase, and pectinesterase genes were downregulated (Table S6, Fig. 7). Upregulation of several genes suggests that these genes may be involved in petal development in hydroponic systems.

Floral organ morphogenesis and growth are also influenced by multiple phytohormones. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, which promote petal and stamen growth in Arabidopsis thaliana [61]. Gibberellic acid and brassinosteroids promote petal development; however, abscisic acid has an antagonistic role in petal development [62, 63]. The expression patterns of phytohormone pathway genes are different under different culture systems. Most DEGs involved in the auxin, jasmonate and brassinosteroid signaling pathways were upregulated (Table S9); however, most DEGs involved in the abscisic acid pathway were downregulated in the hydroponic system (Table S9). This suggests that effects of the hydroponic system on chrysanthemum flower growth may be dependent on phytohormone signaling.

One beneficial effect of hydroponic systems is improved photosynthesis and reduction of the portion of fixed CO2 released into the rooting medium as non-respiratory carbon [64]. The conventional conception of most white or colored flower petals is that they are composed of non-photosynthetic tissues that lack chlorophyll. However, Lysenko and Varduny (2013) found that colored flower petals of Petunia hybrida that lack chlorophyll are capable of light energy storage, as well as usual chlorophyll photosynthesis accompanied by additional ATP synthesis [65]. In this experiment, DEGs involved in photosynthesis pathways exhibited more obvious differences between the two culture systems. DEGs encoding chlorophyll a-b binding protein, dihydrodipicolinate reductase-like protein CRR1, psbP-like protein 2, photosystem II PsbW, psbQ-like protein, PDZ domain, magnesium-chelatase subunit ChlI protein, and magnesium chelatase subunit H of important photosynthesis-related genes were upregulated in flowers grown in the hydroponic system (Table S10). Whether chrysanthemum flowers photosynthesize like leaves requires further research; however, these results suggest that hydroponic culture can reduce the expression of some important photosynthesis-related genes.

Biosynthesis of flavonoids, chlorogenic acids, and other metabolites

Epidemiological studies indicate that chronic inflammation and oxidative stress are critical in neoplastic development [66]. Some currently used synthetic free radical scavengers have demonstrated various side effects [67, 68]. Therefore, functional foods and teas have become a promising source of natural antioxidants [66]. Chrysanthemum has a long history as an herbal tea or medicine for the treatment of various inflammatory diseases. Many studies have shown that chrysanthemum has multiple activities, with antioxidant activity being of particular interest [2,3,4,5,6,7]. Therefore, improving the accumulation and activity of secondary metabolites in chrysanthemum has become a hot topic of research.

Chrysanthemum secondary metabolites, such as flavonoids and chlorogenic acid, are non-enzymatic antioxidants. Flavonoid and chlorogenic acid biosynthetic pathways have largely been characterized [43], and have been shown to affect many conditions, such as high and low mineral nutrition [69,70,71,72], drought [16, 73,74,75], heat [75], cold [76], high salinity [77], and other environmental stresses [75, 78]. In the present study, total flavonoid and chlorogenic acid concentrations in flowers grown in a hydroponic system were higher than those grown in a soil system. Interestingly, transcript levels of the DEGs CHR00004510, CHR00004511, CHR00005671, CHR00018282, CHR00029120, CHR00058078, which encode key genes of the phenylpropanoid pathway, were also upregulated in flowers cultured under a hydroponic system (Fig. 7A).

In the current study, low nitrogen treatment given after the chrysanthemum bud differentiation stage increased total flavonoids, chlorogenic acid contents, and the content of other secondary metabolites without affecting yield. Nitrogen content was lower in the hydroponic system than in the soil system (Figure S4). Our findings are also consistent with previously reported studies [69,70,71]. Soil can absorb and fix nutrients that plants are unable to effectively absorb, or are unable to absorb at a given time. However, hydroponic systems allow plants to rapidly take up nutrients and the nutrient content of plants may be altered by changing the nutrient solutions. This simple and effective method provides us with a new way to cultivate chrysanthemums with high levels of calcium, iron and selenium, among others.

The total flavonoid content of extracts from the chrysanthemum variety “Wuyuanhuang” was 36.75–43.22 mg/g DW (Fig. 2). This was lower than extract of “Kunlun XueJu” (87.2 mg/g DW), which was higher than “Huang Ju”, “Hangbai Ju”, “Tai Ju”, “Gong Ju”, and “Ganye Ju”. Chlorogenic acid content of extracts from the chrysanthemum variety “Wuyuanhuang” was 1.71–2.33 mg/g DW, which was higher than “Kunlun XueJu” and “Ganye Ju” [2]. Thus, C. morifolium ‘Wuyuanhuang’ is a good chrysanthemum tea variety. Agreeing with previous studies, our results indicate that individual chrysanthemum samples and different extraction methods might significantly differ in their flavonoid and chlorogenic acid compositions [2, 4, 6, 7]. Although we did not investigate whether hydroponic systems can affect the antioxidant activity of the flowers, previous studies have demonstrated a significant, positive correlation between the antioxidant activity of C. morifolium flowers and the concentrations of total flavonoids and chlorogenic acid [79].

Transcription factors

Comparative transcriptome analysis revealed a wealth of information. Many important TFs were significantly upregulated, and a few TFs were turned off in chrysanthemum flowers cultivated in a hydroponic system (Fig. 9). Transcription factors make important contributions to the regulation of plant growth and development, and can also influence plant secondary metabolites through orchestrating regulatory networks [33,34,35,36,37,38,39,40,41,42,43,44]. The C2H2 zinc finger transcription factors are reportedly involved in many biological processes related to plant or floral growth and development, hormone signaling, and stress responses in Arabidopsis and chrysanthemum [37, 38, 80]. In the present study, 26 and 7 C2H2 genes were up- and down-regulated, respectively, in flowers grown in a hydroponic system, implying that C2H2 TFs are important for chrysanthemum flower development or the biosynthesis of flavonoids and chlorogenic acid.

MYB and bHLH transcription factors are important regulators involved in controlling plant or flower growth and development, and are involved in secondary metabolites synthesis in plants [81,82,83,84]. Notably, MYB–bHLH–WD40 complexes modulate flavonoid biosynthesis in C. morifolium ‘Chuju’ [44]. In this work, many unigenes homologous to MYB and bHLH were identified. Sixty-three and 18 MYB genes, and 50 and 33 bHLH genes were up- and downregulated in flowers grown under a hydroponic system, respectively. One MYB and two bHLH genes were not expressed in flowers grown in soil. Accordingly, functional characterization of these genes is required under hydroponic conditions.

Comparative transcriptome analysis found one WD40 gene was upregulated in flowers grown in a hydroponic system (Table S7). The FACTOR (TCP20) transcription factor of chrysanthemum is reportedly involved in regulation of petal size by interacting with CmJAZ1-like and inducing downregulation of CmBPE2 gene expression [34]. Transcriptome analysis found three TCP TFs (one CYC4 gene) were upregulated in flowers under hydroponic conditions. In this work (Fig. 9), ERF, Trihelix, WRKY and C3H family members were identified in chrysanthemum, and were upregulated during chrysanthemum flower bud differentiation or development [35,36,37,38,39,40,41]. This implies that these genes may be involved in flower developmental processes, and these TFs should be further studied in chrysanthemum. Overall, TFs were upregulated more than downregulated in flowers grown under a hydroponic system, implying that flowers grown hydroponically have more active genes than those grown in soil. This may be why hydroponically grown chrysanthemum flowers have improved yield and active compounds compared with those grown in soil.

Conclusions

In the present study, the yield of chrysanthemum flowers, and total flavonoids and chlorogenic acid of chrysanthemum tea made from flowers grown in a hydroponic culture system, were higher than those grown in a soil system. Transcriptome analysis indicated that DEGs between flowers grown under these two culture systems were enriched in GO and KEGG pathways relating to the regulation pf flower growth and development, and secondary metabolism. Our results will contribute to knowledge of effective and simple cultivation methods for chrysanthemum tea, and identify genes involved in mechanisms influenced by hydroponic systems for improving chrysanthemum yield and total flavonoids and chlorogenic acid.

Methods

Plant materials

All trials were carried out with chrysanthemum stock plants (‘Wuyuanhuang’) that is a Chinese traditional tea Chrysanthemum. All plant materials were owned from our laboratory, Plant Germplasm Resources and Genetic Engineering, Henan University, for 10 years. Chrysanthemum plating experiment was conducted at the Zhenghzhou A Boluo fertilizer company (113°43′N, 34°76′W) that was our unit from June 1 to November 92,020. The rooted young plants were fixed on the polyethylene lay and floated in nutrient solution for cultivation and growth. For comparison, the same Chrysanthemum plants cultivated in soil as control (Fig.1A, B). The initial physico-chemical properties of soil in this experiment, such as pH (8.1), soil nitrogen concentrations (0.97 g/kg), soil available phosphorus concentrations (7.6 mg/kg), soil rapidly available potassium concentration (106 mg/kg) came from the company farm. The experiments were carried out in outdoor. The nutrient solution composition was as: A Boluo chrysanthemum self-fertilizer (nitrogen, phosphorus, potassium and trace elements) 10 g / 20 L water, pH adjusted as 5.8–6.0, EC was 1.0–1.3 ms/ cm. During the seedling stage, water was added every day to maintain the normal supply (loss of water cause by evaporation due to high temperature) and determine EC weekly. Adding fertilizer adjusted the solution to 1.0–1.3 ms/cm. Nitrogen treatment was stopped when plants entered the full bloom stage. Harvesting took place after a week of nitrogen stoppage. The soil cultivated chrysanthemum plants were treated with the same amount of fertilizer as hydroponic system.

All flowers of each plant were collected and dried at 40–45 °C in a forced-air oven system according to statistic quantity and weight caliber (Fig. 1D, E). The flowers were pulverized and stored at − 25 °C for quality analysis.

Measurement of total flavonoid, chlorogenic acid and water extracts concentration

Total flavonoid was measured as described by Dahui Liu (2010) [70]. Briefly, 1.0 g powered sample of flowers were extracted with 60 mL 70% ethanol in shaker at 60 °C for 30 min,160 r/min speed, then filtered and added 70% ethanol to 100 mL. One milliliter of the ethanol extract was added to the test tube, followed by the addition of 4 mL 60% ethanol and 0.5 mL 5 g/100 mL NaNO2 solution. After 5 min, 0.5 mL 10 g/100 mL Al (NO3)3 solution and 4 mL 10 g/100 mL NaOH were added, respectively. The samples were incubated for 15 min. Then, the absorbance was measured at 510 nm using a spectrophotometer. The content of total flavonoids was calculated as rutin equivalents.

Chlorogenic acid was measured according to the methods described by Jinlong Wan (2019) [85]. A total of 0.05 g of powered sample was immersed in 5 mL of methanol-0.4% phosphoric acid solution. After 24 h, they were sonicated for 30 min and centrifuged for 5 min at 16,000 g. Then the suspension was filtered through a 0.22 μm millipore membrane for HPLC equipped with a ZORBAX Eclipse XDB-C18 column (250 × 4.6 mm, 5 μm; Agilent, Santa Clara, CA, USA). Two solvent systems of the mobile phase: solvent A, acetonitrile/0.4% phosphoric acid (10:90, v:v); solvent B, 100% acetonitrile. The HPLC operating procedures was the same as that described in Jinlong Wan (2019) [85]. Chlorogenic acid concentrations of samples were calculated in relation to standard curves generated with relevant standards.

The water extracts were measured as described by Jianyong Zhang (2010) [86]. Briefly, 1.0 g of powdered sample was immersed in 150 mL hot ddH2O in boiling water bath for 45 min and shake once every 10 min, then vacuum filtered. Weighing chrysanthemum residues that heated in 120 °C dryer for1 h. The above process was repeated until two consecutive weighing of chrysanthemum residue mount difference was less than 0.005 g. The water extracts content (%) = (1-the weight of the last chrysanthemum residue/weight of powdered sample) × 100%.

Measurement of N concentration

Flower samples power was digested in sulfuric acid (H2SO4) and hydrogen peroxide (H2O2), the assay of total N was performed as described previously by Liu Wei (2010) [71].

RNA isolation and sequencing

Total RNA of chrysanthemum flowers extraction, RNA-Seq cDNA library preparation and sequencing were carried out by Shanghai Origingene Bio-pharm Technology Co. Ltd. According to the manufacturer’s protocol, total RNA from chrysanthemum flowers were extracted and purified using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA). RNA concentration was measured by nanophotometer (Implen, Inc., Westlake Village, CA, USA). RNA integrity was assessed through the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Briefly, cDNA library construction was performed with the TruseqTM RNA sample prep Kit for Illumina (NEB, USA). In total six libraries of flowers under two culture system were sequenced on the Illumina Hiseq™ 2500 (Shanghai Origingene Bio-pharm Technology Co. Ltd. Shanghai, China).

Analysis of RNA-sequence data

Using HISAT (Version: v2.1.0) [87, 88] to filter and align the reads, and mapped the clean reads with the chrysanthemum indicum ‘Nankingense’ gene sequence reference datasets (Chrysanthemum_genome_scaffolds_v2.0) [52]. Then, using perl scripts, the transcriptome from all samples were merged to reconstruct a comprehensive transcriptome. Based on final transcriptome, StringTie [89] and Ballgown [90] were used to estimate the expression levels of all transcripts. The differentially expressed genes were selected by R package edgeR [90], the fold differences (> = 2), statistical significance (FDR < 0.05).

Enrichment analysis

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to obtain more detailed information about DEGs in GO terms (http://geneontology.org/) (Gene ontology consortium 2001) and metabolic pathways (https://www.genome.jp/kegg/) [91]. The DEGs in the KEGG pathways and GO analysis were enriched by ClusterProfile R packages [55]. The threshold at padj < 0.01 was determined the significant enrichment of KEGG pathways and GO terms.

Real-time PCR analysis

To validate the gene expression, the extraction of total RNA, cDNA synthesis, and qRT-PCR were performed as previously described [92]. We chose ten DEGs involved in flavonoid synthesis genes, plant hormone signal transduction genes, photosynthesis genes, and the primer sequences were listed in Table S8. We selected chrysanthemum GAPDH (Gene id: CHR00001231) as a reference for gene expression analysis. We calculate the relative expression of mRNA using 2-ΔΔCT equation [93, 94].

Statistical analysis

Using SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA) performed the statistical analysis of the number and dry weight of the chrysanthemum flowers, flavonoids and chlorogenic acid contents, N Concentration of the chrysanthemum flowers, as well as relative expression level and FPKM analysis.

Availability of data and materials

RNA-Seq data generated in the study have been deposited in the National center for Biotechnology Information (NCBI) under the accession codes of Bio Project ID: PRJNA744367 and SRA submission ID: SUB9968352 (https://www.ncbi.nlm.nih.gov/sra/PRJNA744367).

Abbreviations

DEGs:

Differentially expressed genes

GO:

Gene Ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

FPKM:

Reads mapped per 1000 bp per million sequenced reads

ABA:

Abscisic acid

AUX:

Auxin

JA:

Jasmonic acid

BR:

Brassinosteroid

GA:

Gibberellin

ARF:

Auxin response factor

EXP:

Expansin

PE:

Pectin esterase

PG:

Polygalacturonase

PL:

Pectate lyase

XTH:

Xyloglucan endotransglycosylase/hydrolase

XYL:

Xylosidase

TF:

Transcription factor

qRT-PCR:

Quantitative real-time PCR

References

  1. 1.

    Dai SL, Wang WK, Huang JP. Advances of researches on phylogeny of Dendranthema and origin of chrysanthemum. J Beijing Forestry Univ. 2002;24(006):230–4.

    Google Scholar 

  2. 2.

    Du H, Li SS, Wu Q, Ji KX, Wu J, Liu Y, et al. Analysis of active compounds and antioxidant activity assessment of six popular Chinese Juhua teas. Nat Prod Commun. 2015;10(3):495–8.

    PubMed  Google Scholar 

  3. 3.

    Wu TY, Khor TO, Saw CL, Loh SC, Chen AI, Lim SS, et al. Anti-inflammatory/anti-oxidative stress activities and differential regulation of nrf2-mediated genes by non-polar fractions of tea chrysanthemum zawadskii and licorice Glycyrrhiza uralensis. AAPS J. 2011;13(1):1–13.

    PubMed  Google Scholar 

  4. 4.

    Li YF, Yang PY, Luo YH, Gao BY, Sun JH, Lu WY, et al. Chemical compositions of chrysanthemum teas and their anti-inflammatory and antioxidant properties. Food Chem. 2019;15(286):8–16.

    Google Scholar 

  5. 5.

    Chen LX, Hu DJ, Lam SC, Ge L, Wu D, Zhao J, et al. Comparison of antioxidant activities of different parts from snow chrysanthemum (Coreopsis tinctoria Nutt.) and identification of their natural antioxidants using high performance liquid chromatography coupled with diode array detection and mass spectrometry and 2,2 -azinobis (3-ethylbenzthiazoline-sulfonic acid) diammonium salt-based assay. J Chromatogr A. 2016;8(1428):134–42.

    Google Scholar 

  6. 6.

    Liu QJ, Liu HF, Yuan ZL, Wei DW, Ye YZ. Evaluation of antioxidant activity of chrysanthemum extracts and tea beverages by gold nanoparticles-based assay. Colloids Surf B Biointerfaces. 2012;1(92):348–52.

    Google Scholar 

  7. 7.

    Jeong SC, Kim SM, Jeong YT, Song CH. Hepatoprotective effect of water extract from Chrysanthemum indicum L. flower. Chin Med. 2013;8(1):7.

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Liang NJ, Kitts DD. Role of chlorogenic acids in controlling oxidative and inflammatory stress conditions. Nutrients. 2015;8(1):16.

    PubMed Central  Google Scholar 

  9. 9.

    Xiao X, Zhu W, Du C, Shi YD, Wang JF. Effect of crop rotation and biological manure on quality and yield of "Chuju" Chrysanthemum morifolium and continuous cropping soil enzyme activities. Zhong Yao Cai. 2015;38(5):889–93.

    CAS  PubMed  Google Scholar 

  10. 10.

    Song AP, Zhao S, Chen SS, Jiang JF, Chen S, Li HY, Chen Y, Chen X, Fang WM, Chen FD. The abundance and diversity of soil fungi in continuously monocropped chrysanthemum. TSWJ. 2013;1:632920.

  11. 11.

    Wang JF, Zhou Y, Gao Q, Zhang P, Xiao X, Chen SY. Isolation, identification and variation characteristics in soil of a continuous cropping Chuzhou chrysanthemum. J Biol. 2011;28(6):46–8.

    Google Scholar 

  12. 12.

    Feng XY, Fang WM, Chen FD, Guan ZY, Jiang JF. The breeding of Chrysanthemum morifolium for tea and medicine. Zhong Yao Cai. 2017;40(2):258–63.

    Google Scholar 

  13. 13.

    Chen X, Zhao S, Yao JJ, Ye YP, Song AP, Chen FD, et al. Effects of bio-organic fertilizer and fungicide application on continuous cropping obstacles of cut chrysanthemum. Ying Yong Sheng Tai Xue Bao. 2015;26(4):1231–6.

    PubMed  Google Scholar 

  14. 14.

    Azeezahmed SK, Dubey RK, Kukal SS, Sethi VP. Effect of different nitrogen-potassium concentrations on growth and flowering of chrysanthemum in a drip hydroponic system. J Plant Nutr. 2016;39(13):1891–8.

    CAS  Google Scholar 

  15. 15.

    Prasanna R, Saxena G, Singh B, Ranjan K, Buddhadeo R, Velmourougane K, et al. Mode of application influences the biofertilizing efficacy of cyanobacterial biofilm formulations in chrysanthemum varieties under protected cultivation. Open Agriculture. 2018;3:478–89.

    Google Scholar 

  16. 16.

    Gharibi S, Tabatabaei BE, Saeidi G, Talebi M, Matkowski A. The effect of drought stress on polyphenolic compounds and expression of flavonoid biosynthesis related genes in Achillea pachycephala Rech.f. Phytochemistry. 2019;162:90–8.

    CAS  PubMed  Google Scholar 

  17. 17.

    Gharibi S, Tabatabaei BE, Saeidi G, Talebi M, Matkowski A. The effect of drought stress on polyphenolic compounds and expression of flavonoid biosynthesis related genes in Achillea pachycephala Rech. f. Phytochemistry. 2019;(162):90-8.

  18. 18.

    Hill WA, Mortley DG, MacKowiak CL, Loretan PA, Tibbitts TW, Wheeler RM, et al. Growing root, tuber and nut crops hydroponically for CELSS. Adv Space Res. 1992;12:125–31.

    CAS  PubMed  Google Scholar 

  19. 19.

    Kawasaki A, Okada S, Zhang CY, Delhaize E, Mathesius U, Richardson AE, et al. A sterile hydroponic system for characterising root exudates from specifc root types and whole-root systems of large crop plants. Plant Methods. 2018;14:114.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Miller MH, Walker GK, Tollenaar M, Alexander KG. Growth and yield of maize (Zea mays L.) grown outdoors hydroponically and in soil. Can J Soil Sci. 1989;692:295–302.

    Google Scholar 

  21. 21.

    Brentlinger D. New trends in hydroponic crop production in the U. S Acta Hortic. 2007;742:31–4.

    Google Scholar 

  22. 22.

    Brentlinger, D. New trends in hydroponic crop production in the U. S. Acta Hortic. 2007;(742):31-4.

  23. 23.

    Chandra S, Khan S, Avula B, Lata H, Yang MH, ElSohly MA, et al. Assessment of total phenolic and flavonoid content, antioxidant properties, and yield of aeroponically and conventionally grown leafy vegetables and fruit crops: a comparative study. Evid Based Complement Alternat Med. 2014:253875.

  24. 24.

    Chang DC, Park CS, Kim SY, Lee YB. Growth and tuberization of hydroponically grown potatoes. Potato Res. 2012;55:69–81.

    Google Scholar 

  25. 25.

    Barbosa GL, Gadelha FD, Kublik N, Proctor A, Reichelm L, Weissinger E, et al. Comparison of land, water, and energy requirements of lettuce grown using hydroponic vs. conventional agricultural methods. Int. J. Environ. Res. Public Health. 2015;12:6879–91.

    Google Scholar 

  26. 26.

    Da Silva MG, Soares TM, Gheyi HR, Oliveira IS, Da Silva Filho JA, Do Carmo FF. Frequency of recirculation of nutrient solution in hydroponic cultivation of coriander with brackish water. R Bras Eng Agríc Ambiental. 2016;20(5):447–54.

    Google Scholar 

  27. 27.

    Sharma N, Acharya S, Kumar K, Singh N, Chaurasia OP. Hydroponics as an advanced technique for vegetable production: An overview. J Soil Water Conserv. 2018;17(4):364–71.

    Google Scholar 

  28. 28.

    Rolot JL, Seutin H. Soilless production of potato minitubers using a hydroponic technique. Potato Res. 1999;42:457–69.

    Google Scholar 

  29. 29.

    Schmautz Z, Loeu F, Liebisch F, Graber A, Mathis A, Bulc TGG, et al. Tomato productivity and quality in aquaponics: comparison of three hydroponic methods. Water. 2016;8:533.

    Google Scholar 

  30. 30.

    Barbosa JG, Barbosa MS, Almeida DB, Sá PG, Finger FL, Martinez HEP, et al. Production and postharvest quality of chrysanthemum flowers grown in hydroponic system under different N:K ratios. Acta Hortic. 2015;1060(42):281–7.

    Google Scholar 

  31. 31.

    Shan B, Cai YZ, Brooks JD, Corke H. The in vitro antibacterial activity of dietary spice and medicinal herb extracts. Int J Food Microbiol. 2007;117(1):112–9.

    CAS  PubMed  Google Scholar 

  32. 32.

    Xie YY, Yuan D, Yang JY, Wang LH, Wu CF. Cytotoxic activity of flavonoids from the flowers of chrysanthemum morifolium on human colon cancer colon 205 cell. J Asian Nat Prod Res. 2009;11(9):771–8.

    CAS  PubMed  Google Scholar 

  33. 33.

    Huang D, Li XW, Sun M, Zhang HT, Chen TR, Wang J, et al. Identification and characterization of CYC-like genes in regulation of ray floret development in chrysanthemum morifolium. Front Plant Sci. 2016;7:1633.

    PubMed  PubMed Central  Google Scholar 

  34. 34.

    Wang JJ, Guan YX, Ding L, Li PR, Zhao WQ, Jiang JF, et al. The CmTCP20 gene regulates petal elongation growth in chrysanthemum morifolium. Plant Sci. 2019;280:248–57.

    CAS  PubMed  Google Scholar 

  35. 35.

    Pu Y, Huang H, Wen XH, Lu CF, Zhang BH, Gu XQ, et al. Comprehensive transcriptomic analysis provides new insights into the mechanism of ray floret morphogenesis in chrysanthemum. BMC Genomics. 2020;21:728.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Hong Y, Tang XJ, Huang H, Zhang Y, Dai SL. Transcriptomic analyses reveal species-specific light-induced anthocyanin biosynthesis in chrysanthemum. BMC Genomics. 2015;16:202.

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Wang Y, Huang H, Ma YP, Fu JX, Wang LL, Dai SL. Construction and de novo characterization of a transcriptome of Chrysanthemum lavandulifolium: analysis of gene expression patterns in floral bud emergence. Plant Cell Tissue Organ Cult. 2014;116:297–309.

    CAS  Google Scholar 

  38. 38.

    Ren LP, Liu T, Chen Y, Sun J, Cao JJ, Dong B, et al. Transcriptomic analysis of differentially expressed genes in the floral transition of the summer flowering chrysanthemum. BMC Genomics. 2016;17:673.

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Wang JJ, Wang HB, Ding L, Song AP, Shen F, Jiang JF, et al. Transcriptomic and hormone analyses reveal mechanisms underlying petal elongation in chrysanthemum morifolium ‘Jinba’. Plant Mol Biol. 2017;93:593–606.

    CAS  PubMed  Google Scholar 

  40. 40.

    Liu H, Du DL, Pan HT, Cheng TR, Wang J, Zhang QX. Whole-transcriptome analysis of differetially expressed genes in the vegetative buds, floral buds and buds of chrysanthemum morifolium. PLoS One. 2015;10:e0128009.

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Fu JX, Zhang C, Liu YC, Pang TH, Dong B, Gao XY, et al. Transcriptomic analysis of flower opening response to relatively low temperatures in Osmanthus fragrans. BMC Plant Biol. 2020;20:337.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Ma J, Li Z, Wang B, Sui S, Li M. Cloning of an expansin gene from Chimonathus praecox flowers and its expression in flowers treated with ethephon or 1-methylcyclopropene. Hortscience. 2012;47(10):1472–7.

    CAS  Google Scholar 

  43. 43.

    Falcone Ferreyra ML, Rius SP, Casati P. Flavonoids: biosynthesis, biological functions, and biotechnological applications. Front Plant Sci. 2012;3:222.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Yue JY, Zhu CX, Zhou Y, Niu XL, Miao M, Tang XF, et al. Transcriptome analysis of differentially expressed unigenes involved in flavonoid biosynthesis during flower development of Chrysanthemum morifolium ‘Chuju’. Sci Rep. 2018;8:13414.

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Lu CF, Pu Y, Liu YT, Li YJ, Qu JP, Huang H, et al. Comparative transcriptomics and weighted gene co-expression correlation network analysis (WGCNA) reveal potential regulation mechanism of carotenoid accumulation in Chrysanthemum × morifolium. Plant Physiol Biochem. 2019;142:415–28.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Dong W, Li MM, Li ZA, Li SL, Zhu Y, Xu H, et al. Transcriptome analysis of the molecular mechanism of Chrysanthemum flower color change under short-day photoperiods. Plant Physiol Biochem. 2020;146:315–28.

    PubMed  Google Scholar 

  47. 47.

    Huang H, Wang Y, Wang SL, Wu X, Yang K, Niu YJ, et al. Transcriptome-wide survey and expression analysis of stress-responsive NAC genes in Chrysanthemum lavandulifolium. Plant Sci. 2012;193-194:18–27.

    CAS  PubMed  Google Scholar 

  48. 48.

    Cheng PL, Gao JJ, Feng YT, Zhang ZX, Liu YN, Fang WM, et al. The chrysanthemum leaf and root transcript profiling in response to salinity stress. Gene. 2018;674:161–9.

    CAS  PubMed  Google Scholar 

  49. 49.

    Cheng PL, Gao JJ, Feng YT, Zhang ZX, Liu YN, Fang WM, Chen SM, Chen FD, Jiang JF. The chrysanthemum leaf and root transcript profiling in response to salinity stress. Gene. 2018;(674):161-9.

  50. 50.

    Sun J, Ren LP, Cheng Y, Gao JJ, Dong B, Chen SM, et al. Identification of differentially expressed genes in Chrysanthemum nankingense (Asteraceae) under heat stress by RNA Seq. Gene. 2014;552:59–66.

    CAS  PubMed  Google Scholar 

  51. 51.

    Ren LP, Sun JP, Chen SM, Gao JJ, Dong B, Liu YN, Xia XL, Wang YJ, Liao Y, Teng NJ, Fang WM, Guan ZY, Chen FAD, Jiang JF. A transcriptomic analysis of Chrysanthemum nankingense provides insights into the basis of low temperature tolerance. BMC Genomics. 2014, 15:844.

  52. 52.

    Song C, Liu YF, Song AP, Dong GQ, Zhao HB, Sun W, et al. The Chrysanthemum nankingense genome provides insights into the evolution and diversification of chrysanthemum flowers and medicinal traits. Mol Plant. 2018;11(12):1482–91.

    CAS  PubMed  Google Scholar 

  53. 53.

    Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28:511–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Boil. 2014;15:550.

    Google Scholar 

  55. 55.

    Yu GH, Wang LG, Han YY, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. Omics. 2012;16:284–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Cosgrove DJ. Wall structure and wall loosening. A look backwards and forwards. Plant Physiol. 2001;125(1):131–4.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Zenoni S, Reale L, Tornielli GB, Lanfaloni L, Porceddu A, Ferrarini A, et al. Downregulation of the Petunia hybrida alpha-expansin gene PhEXP1 reduces the amount of crystalline cellulose in cell walls and leads to phenotypic changes in petal limbs. Plant Cell. 2004;16(2):295–308.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    O'Donoghue EM, Somerfield SD, Heyes JA. Organization of cell walls in Sandersonia aurantiaca floral tissue. J Exp Bot. 2002;53(368):513–23.

    CAS  PubMed  Google Scholar 

  59. 59.

    Gookin TE, Hunter DA, Reid MS. Temporal analysis of alpha and betaexpansin expression during floral opening and senescence. Plant Sci. 2003;164(5):769–81.

    CAS  Google Scholar 

  60. 60.

    Harada T, Torii Y, Morita S, Onodera R, Hara Y, Yokoyama R, et al. Cloning, characterization, and expression of xyloglucan endotransglucosylase/hydrolase and expansin genes associated with petal growth and development during carnation flower opening. J Exp Bot. 2011;62(2):815–23.

    CAS  PubMed  Google Scholar 

  61. 61.

    Reeves PH, Ellis CM, Ploense SE, Wu MF, Yadav V, Tholl D, et al. A regulatory network for coordinated flower maturation. PLoS Genet. 2012;8(2):e1002506.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Huang G, Han M, Yao W, Wang Y. Transcriptome analysis reveals the regulation of brassinosteroids on petal growth in Gerbera hybrida. Peer J. 2017;5(5):e3382.

    PubMed  PubMed Central  Google Scholar 

  63. 63.

    Hedden P, Thomas SG. Gibberellin biosynthesis and its regulation. Biochem J. 2012;444(1):11–25.

    CAS  PubMed  Google Scholar 

  64. 64.

    Smucker AJ. Carbon utilization and losses by plant root systems; 1982. p. 27–46. in S.A

    Google Scholar 

  65. 65.

    Lysenko V, Varduny T. Anthocyanin-dependent anoxygenic photosynthesis in coloured flower petals? Sci Rep. 2013;3:3373.

    PubMed  PubMed Central  Google Scholar 

  66. 66.

    Clevers H. At the crossroads of inflammation and cancer. Cell. 2004;118(6):671–4.

    CAS  PubMed  Google Scholar 

  67. 67.

    Blaszczyk A, Skolimowski J. Comparative analysis of cytotoxic, genotoxicand antioxidant effects of 2,2,4,7-tetramethyl-1,2,3,4-tetrahydroquinolineand ethoxyquin on human lymphocytes. Chem Biol Interact. 2006;162:70–80.

    CAS  PubMed  Google Scholar 

  68. 68.

    Błaszczyk A, Skolimowski J, Materac A. Genotoxic and antioxidant activities ofethoxyquin salts evaluated by the comet assay. Chem Biol Interact. 2006;162:268–73.

    PubMed  Google Scholar 

  69. 69.

    Fernandes L, Casal S, Pereira JA, Saraiva JA, Ramalhosa E. Edible flowers: a review of the nutritional, antioxidant, antimicrobial properties and effects on human health. J Food Composi Anal. 2017;60:38–50.

    CAS  Google Scholar 

  70. 70.

    Liu DH, Liu W, Zhu DW, Geng MJ, Zhou WB, Yang TW. Nitrogen effects on total flavonoids, chlorogenic acid, and antioxidant activity of the medicinal plant Chrysanthemum morifolium. J Plant Nutr Soil Sci. 2010;173:268–74.

    CAS  Google Scholar 

  71. 71.

    Liu W, Zhu DW, Liu DH, Geng MJ, Zhou WB, Mi WJ, et al. Influence of nitrogen on the primary and secondary metabolism and synthesis of flavonoids in chrysanthemum morifolium ramat. J Plant Nutr. 2010;33:240–54.

    Google Scholar 

  72. 72.

    Liu W, Zhu DW, Liu DH, Zhou WB, Yang TW, Geng MJ. Influence of potassium deficiency on flower yield and flavonoid metabolism in leaves of chrysanthemum morifolium ramat. J Plant Nutr. 2011;34:1905–18.

    CAS  Google Scholar 

  73. 73.

    Hodaei M, Rahimmalek M, Arzani A, Talebi M. The effect of water stress on phytochemical accumulation, bioactive compounds and expression of key genes involved in flavonoid biosynthesis in Chrysanthemum morifolium L. Ind Crop Prod. 2018;120:295–304.

    CAS  Google Scholar 

  74. 74.

    Hodaei M, Rahimmalek M, Arzani A, Talebi M. The effect of water stress on phytochemical accumulation, bioactive compounds and expression of key genes involved in flavonoid biosynthesis in Chrysanthemum morifolium L. Ind. Crops Prod. 2018;(120):295-304.

  75. 75.

    Tattini M, Galardi C, Pinelli P, Massai R, Remorini D, Agati G. Differential accumulation of flavonoids and hydroxycinnamates in leaves of Ligustrum vulgare under excess light and drought stress. New Phytol. 2004;163:547–61.

    CAS  PubMed  Google Scholar 

  76. 76.

    Korn M, Peterek S, Mock HP, Heyer AG, Hincha DK. Heterosis in the freezing tolerance, and sugar and flavonoid contents of crosses between Arabidopsis thaliana accessions of widely varying freezing tolerance. Plant Cell Environ. 2008;31:813–27.

    CAS  PubMed  PubMed Central  Google Scholar 

  77. 77.

    Walia H, Wilson C, Condamine P, Liu X, Ismail AM, Zeng L, et al. Comparative transcriptional profiling of two contrasting rice genotypes under salinity stress during the vegetative growth stage. Plant Physiol. 2005;139:822–35.

    CAS  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Li J, Ou-Lee TM, Raba R, Amundson RG, Last RL. Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell. 1993;5:171–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Wang S, Hao L, Zhu J, Wang Z, Zhang X, Song X. Comparative evaluation of Chrysanthemum Flos from different origins by HPLC-DAD-MSn and relative response factors. Food Anal Methods. 1993;8:40–51.

    Google Scholar 

  80. 80.

    Prunet N, Yang WB, Das P, Meyerowitz EM, Jack TP. SUPERMAN prevents class B gene expression and promotes stem cell termination in the fourth whorl of Arabidopsis thaliana flowers. Proc Natl Acad Sci U S A. 2017;114(27):7166–71.

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81.

    An XH, Tian Y, Chen KQ, Liu XJ, Liu DD, Xie XB, et al. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant Cell Physiol. 2015;56(4):650–62.

    CAS  PubMed  Google Scholar 

  82. 82.

    Zhang J, Xu HF, Wang N, Jiang SH, Fang HC, Zhang ZY, et al. The ethylene response factor MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis in apple. Plant Mol Biol. 2018;98:205–18.

    CAS  PubMed  Google Scholar 

  83. 83.

    Li ST. Transcriptional control of flavonoid biosynthesis fine-tuning of the MYB-bHLH-WD40 (MBW) complex. Plant Signal Behav. 2014;9(1):e27522.

    PubMed  PubMed Central  Google Scholar 

  84. 84.

    Liu JY, Osbourn A, Ma PD. MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Trends Plant Sci. 2019;24(3:275–89.

    Google Scholar 

  85. 85.

    Wan JL, Huang B, Yu H, Peng SL. Reassociation of an invasive plant with its specialist herbivore provides a test of the shifting defence hypothesis. J Ecol. 2019;107:361–71.

    Google Scholar 

  86. 86.

    Zhang JY, Jiang HY, Yuan XY, Yao Y, Liu HY, Cui HC. Content analysis of the flavonoids, polyphenols and water-extracts in Chrysanthemum. Chin Agric Sci Bull. 2010;26(9):102–5.

    Google Scholar 

  87. 87.

    Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  88. 88.

    Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015;33(3):290–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  89. 89.

    Frazee AC, Pertea G, Jaffe AE, Langmead B, Salzberg SL, Leek JT. Ballgown bridges the gap between transcriptome assembly and expression analysis. Nat Biotechnol. 2015;33(3):243–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  90. 90.

    Robinson MD, Mccarthy DJ, Smyth GK. Edger: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139–40.

    CAS  PubMed  PubMed Central  Google Scholar 

  91. 91.

    Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M. KEGG for integration and interpretation of largescale molecular data sets. Nucleic Acids Res. 2012;40:D109–14.

    CAS  PubMed  Google Scholar 

  92. 92.

    Kang DR, Li ZA, Li MM, Li J, Liu YH, Ai PH, et al. Dynamic changes of DNA methyltransferase and demethylase gene expression during Chrysanthemum × morifolium flower induction and development. Eur J Hortic Sci. 2021;86(2):122–9.

    Google Scholar 

  93. 93.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods (San Diego, Calif). 2001;25(4):402–8.

    CAS  Google Scholar 

  94. 94.

    Zhang N, He ZJ, He SY, Jing P. Insights into the importance of dietary chrysanthemum flower (Chrysanthemum morifolium cv. Hangju)-wolfberry (Lycium barbarum fruit) combination in antioxidant and anti-inflammatory properties. Food Res Int. 2019;116:810–8.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This research was supported by National Key Research and Development Program (2020YFD1000401), Project of Science and Technology Department of KaiFeng (2019009 ZK), Henan Science and Technology Research Project (162300410250). The funding organizations provided the financial support and were involved in the design of this study, analysis of the data, writing of the manuscript.

Author information

Affiliations

Authors

Contributions

WZ designed the research. AP and LX drafted the manuscript. LZ, KD and Muhammad Ayoub Khan performed the transcriptome data processing and analysis. LH and SM performed total flavonoid, chlorogenic acid, the water extracts and nitrogen analysis. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Zicheng Wang.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Figure S1.

Pearson correlation coefficients among 3 biological replications from chrysanthemum flowers under the soil system (Flower-CK-1, Flower-CK-2 and Flower-CK-3) and hydroponic system (Flower-SP-1, Flower-SP-2 and Flower-SP-3). The numbers in the scale bar stand for correlation coefficients. Figure S2. Principal component analysis among 3 biological replications from flowers under soil and hydroponic system. Figure S3. Difference of total nitrogen content in chrysanthemum flower under soil and hydroponic system. *P < 0.05, **P < 0.01.

Additional file 2: Table S1.

GO enrichment of chrysanthemum flowers. Table S2. KEGG pathways enriched in the chrysanthemum flowers. Table S3. List of flavonoid biosynthesis-related DEGs. Table S4. List of Phenylpropanoid biosynthesis-related DEGs. Table S5. List of fatty acid and unsaturated fatty acid biosynthesis-related DEGs. Table S6. List of cell wall metabolism-related DEGs. Table S7. List of TF-related DEGs. Table S8. List of the quantitative primers for qRT-PCR in this study. Table S9. List of hormone-related DEGs. Table S10. List of photosynthesis-related DEGs.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ai, P., Liu, X., Li, Z. et al. Comparison of chrysanthemum flowers grown under hydroponic and soil-based systems: yield and transcriptome analysis. BMC Plant Biol 21, 517 (2021). https://doi.org/10.1186/s12870-021-03255-4

Download citation

Keywords

  • Chrysanthemum tea
  • Hydroponic and soil system
  • Quality
  • RNA-seq analysis