Effect of soil moisture regimes in the early flowering stage on inflorescence morphology and medicinal ingredients of Chrysanthemum morifolium Ramat. Cv. ‘Hangju’

Scientia Horticulturae 260 (2020) 108849

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Effect of soil moisture regimes in the early flowering stage on inflorescence morphology and medicinal ingredients of Chrysanthemum morifolium Ramat. Cv. ‘Hangju’

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Wenyan Zhang, Tao Wang, Qiaosheng Guo , Qingjun Zou, Feng Yang, Dehua Lu, Jing Liu Nanjing Agricultural University, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Water stress Flower-bud differentiation CYCLOIDEA TCP Flower symmetry Flavonoids

The quality of the traditional Chinese medicine (TCM) is closely related to the climatic conditions of its medicinal plant’s habitats. When Chrysanthemum morifolium Ramat. cv. ‘Hangju’ (Hj) is in the period of flower-bud differentiation, the rainy season also prevails in its original places. In this study, we set three groups of soil moisture regimes (SMR) corresponding to 35%–40% (S1), 65%–70% (S2), 95%–100% (S3) of soil water holding capacity (WHC) and we found for the first time that the effects of different SMR on the inflorescence of Hj in the early flowering stage. With the increase of SMR content, both ray florets (RFs) and disc florets (DFs) are inhibited on inflorescence morphology, especially for DFs. In addition, a CYC2c orthologous gene in “Zaoyang” (ZY, a cultivar of Hj) was identified and sequenced, the phylogenetic analysis showed that this CYCLOIDEA gene in Asteraceae shared duplications which were well-conserved and lineage-specific. Expression analysis showed that HjCYC2c was mainly expressed in RFs, and high SMR condition led to up-regulation. At the same time, our study suggests that FLS gene is a strong candidate as a key factor in Hj for further interaction studies between flavonoids biosynthesis and regulatory factors of floral symmetry. The determination results of medicinal ingredients showed that DFs were more quickly to respond to the SMR variation and produced more medicinal ingredients compared with RFs.

1. Introduction In the traditional Chinese medicine (TCM) theory, the theory of medicine is closely related to the efficacy of compatibility. There is a direct relationship between the original plant traits of the medicinal materials and the efficacy of TCM. At present, a large number of studies are devoted to exploring the natural attribute characteristics of traditional original plants of TCM and the influence of external environment on the quality of medicinal materials. In many geo-authentic habitats of TCM, its original plants was inevitably encountered some stress conditions. The so-called "stress", compared to the normal growth conditions, the influence of excessive climatic factor on plants means that an urgent signal was received in the secondary metabolism pathway, perhaps an opportunity for medicinal plants. Chrysanthemum morifolium Ramat. cv. Hj is one of the original plant series of TCM "Ju Hua", and its inflorescence is composed of peripheral whorls of ray florets (RFs) and multiple whorls of central disc florets

(DFs), which together constitute the quality basis of this medicinal plant. From the perspective of flower symmetry, flowers can be grouped into three categories: asymmetrical, bilateral and radial (Spencer and Kim, 2018). In Hj, RF lacks functional stamens and is bilateral, while DF is hermaphroditic and radial. Dorsoventral asymmetry in flowers is thought to have evolved many times from a radially symmetrical ancestral condition (Cubas, 2004). Studies have shown that CYC homologous genes are one of the major genes controlling the formation of flower symmetry in plants. And, a majority of the shifts from zygomorphy to actinomorphy appear to have entailed a reduction in petal number and flower size, implying a mechanism other than loss of CYCLOIDEA function (Donoghue et al., 1998). There are two types of TCP protein, based on differences within their TCP domains: class I and class II, CYC clade belongs to class II (Martin-Trillo and Cubas, 2010). Within the CYC/TB1 clade, there are three major subclades within core eudicots, which we call CYC1, CYC2, and CYC3; The CYC2 clade contains the greatest number of additional

Abbreviations: WF, whole floret; DF, disc floret; RF, ray floret; ZY, Zaoyang; Hj, Hangju; SMR, soil moisture regimes; TCM, traditional Chinese medicine; WHC, soil water holding capacity ⁎ Corresponding author. E-mail address: [email protected] (Q. Guo). https://doi.org/10.1016/j.scienta.2019.108849 Received 13 August 2019; Received in revised form 10 September 2019; Accepted 11 September 2019 Available online 04 October 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

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floral symmetry and lay a foundation for subsequent molecular experiments.

duplications (Howarth and Donoghue, 2006). For example, there are six CYC2-like members in Asteraceae; they are results of five duplication events starting from a single-copy gene in the common ancestor of the Goodeniaceae-Calyceraceae-Asteraceae group and completing before the divergence of the subfamily Carduoideae of Asteraceae (Chen et al., 2018). In Gerbera hybrid, ectopic activation of GhCYC5 increases flower density in the inflorescence, suggesting that GhCYC5 may promote the flower initiation rate during expansion of the capitulum, however, the organization of the capitulum was not affected, and its appearance did not differ from the wild type (Juntheikki-Palovaara et al., 2014). Instead of regulating the dorsoventral symmetry of individual flowers, GhCYC2 participates in the control of the identity and radial extent of flower types in Gerbera inflorescences. Moreover, GhCYC2 plays an important and unique role in organ fusion that further differentiates Gerbera flower types (Broholm et al., 2008). Furthermore, from previous studies we realized that the key role of CYC2c in regulating the RFs. Double-flowered sunflower mutants (in which DFs develop bilateral symmetry), an insertion into the promoter region of a CYCLOIDEA (CYC)-like gene (HaCYC2c) that is normally expressed specifically in WT rays is instead expressed throughout the inflorescence, presumably resulting in the observed loss of actinomorphy (Chapman et al., 2012). Also, the results in Asteraceae indicated that CYC2c is involved in regulating ray floret identity and played an essential role (Huang et al., 2016; Chen et al., 2018). Hence, we cloned CYC2c of Hj in this study and explored its expression patterns which may used for further function researches. Chrysanthemum morifolium Ramat. (Compositae), distinguished from ornamental plants, is listed in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Committee, 2015). The distribution of Hj's production areas in China is mainly dominated by Tongxiang area of the original production area and presents a trend from south to north (dimension from low to high). The emerging production areas include Sheyang of Jiangsu province, Macheng of Hubei province, and Ruicheng of Shanxi province. In addition, the cultivar of ZY is the main selection of Hj in the cultivation (the variety has passed the identification of crop varieties in Zhejiang Province). The previous research of our group found that the number and rounds of RFs in the origin are significantly higher than other introduced areas, and the diameter of inflorescence also became larger with the increase of latitude (Wang et al., 2012). At the same time, the internal quality also showed various kinds as seen that the difference in the content of the medicinal ingredients. A comprehensive comparison of the climatic factors found in the various production areas of Hj, the significant difference is mainly reflected in the annual precipitation. In addition, when Hj is in its flower-bud differentiation period, however, the rainy season exactly prevails in its original places. Furthermore, in our previous studies and the data of transcriptome sequencing of ZY under flooding stress during the period of flower-bud differentiation, we found a increase in expression of genes related to the flavonoid synthesis pathway (Zou et al., 2018). Therefore, we speculated that excessive moisture conditions may promote the increase of active ingredients. Taken together, this study speculates that water factor may be one of the important factors leading to differences in the external flower-shaped traits and intrinsic medicinal components in the flower heads of different cultivars. In this study, based on the changes of the heterogeneity of quality such as inflorescence morphology and medicinal ingredients in Hj that found in the natural environment, we firstly selected HjCYC2c gene from the perspective of flower symmetry, cloned and conducted gene expression research, expecting to find this regulatory gene’s differential expression. Then, SMR treatments were carried out, expecting to explore the influence of water in the early flowering stage on inflorescence morphology and medicinal ingredients, study the relationship between the original plant habitats and the quality of the medicinal materials. At last, we conducted the gene expression research and the determination of medicinal ingredients to explore the relationship between flavonoids biosynthesis and regulatory factors of

2. Materials and methods 2.1. Plant materials and growth conditions The experimental materials were collected from the medicinal chrysanthemum germplasm of Nanjing Agricultural University, and identified as the medicinal chrysanthemum cultivation type ZY by Dr. Qiao-sheng Guo of Traditional Chinese Medicine Research Institute of Nanjing Agricultural University. At the beginning of April 2018, 100 healthy and growing chrysanthemum seedlings were selected for planting in the plastic flower pots (inner diameter 21 cm, height 17 cm) and grew in the greenhouse of Nanjing Agricultural University, Nanjing, China (118.5 °E, 32.02 °N). The climatic conditions of the greenhouse remained stable at 25/18 °C (day/night temperature), maximum irradiance of 1000–1200 μmol (photon) m−2∙s-1, and a mean relative humidity of 60%. The experimental soil was 1:1 (volume ratio) of nutrient soil and garden soil, and the weight of the soil in each pot was 3 kg. The soil water holding capacity (WHC) was 36.13% measured by cutting ring method. In August 2018, we selected 60 well-grown pots and divided into three groups averagely and randomly before the early flowering stage, and these plants has taken roots adequately. The whole period of treatment lasted for 62 days and we let these plants continue to grow after treatment for sample collection. During the flower-bud differentiation period (Fig. 1), according to the previous research schemes, we set flowering period I (the RFs and DFs both were not open) as the beginning of water treatment, with different SMR corresponding to 35%–40% (S1), 65% 70% (S2), 95%–100% (S3) of WHC, 20 replicative pots per treatment. Moisture control is carried out by weighing and hydrating method. During the treatment period, the water was replenished once a day from 17:00 to 18:00, and the potting position was randomly changed every week to reduce the influence of environmental unevenness on its growth. 2.2. Botanical traits statistics 15 representative plants were selected for each treatment when the DFs was opened 50% and the RFs was opened 70% (Wang et al., 2014). We measured the following botanical traits items by ruler and vernier caliper: the diameter of the inflorescence, the diameter of all DFs, the length and width of the corolla tube of external RFs, the length and width of the corolla tube of innermost RFs. Then we counted the number of RFs and DFs, the round of RFs, and calculated the proportion of the number of RFs in the total florets. Above all the measurement indexes were statistics based on a single inflorescence. Means were separated using one-way ANOVA analysis with Duncan test at P = 0.05. The results were presented as the means ± standard deviation (s.d.) of all biological repeats. Statistical analysis was conducted using SPSS 25.0 after testing the homogeneity of variance. 2.3. Isolation of HjCYC2c genes Total RNA was extracted from the young inflorescence of ZY, with TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan) according to protocol-1 for simple plant tissue and evaluated by 1% agarose gel electrophoresis and nucleic acid detector. The electrophoresis bands with clear, non-degradable and A260/A280 for total RNA is in 1.8 to 2.0, which were selected for reverse transcription. The first strand of cDNA was reverse transcribed using the Random 6 mers primer in the PrimeScript II 1 st Strand cDNA Synthesis Kit (TaKaRa, Japan). Primers 5 software was used to design primers from both ends of the sequence ORF region, and the full-length open reading frames of HjCYC2c were cloned from Hj by RT-PCR. The PCR reaction was carried out using 2

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Fig. 1. Different capitulum development stages of Hangju and flower morphologies, bars =1 mm (A–D), 5 mm (E–H). (A–E) Different development stages from flower-bud differentiation period before blossom. (I) Inflorescence morphologies of Hangju. (J–L) Deformed flower morphologies of Hangju under S3 treatment.

Supplementary Table S1. Their efficiency was analyzed to ensure they were close to 100%. The PCR procedure was conducted with an initial step of 95 °C for 30 s, then followed by 40 cycles: 10 s at 95 °C, 30 s at 60 °C. Finally, at 72 °C for 30 s. The melting curve was 15 s at 95 °C, 60 s at 60 °C, 15 s at 95 °C, and repeated 3 times. The product specificity of each primer pair was verified by melting curve analysis. In this experiment, the relative expression level was calculated using the 2−ΔΔCt method, and the β-actin gene was used as an endogenous control (Table S1). Statistical analysis and graphs were conducted in GraphPad Prism 7 software using t-test with unpaired comparison.

Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China). The reaction procedure was: pre-denaturation at 95 °C for 3 min, then followed by 35 cycles: 95 °C for 15 s, 55 °C for 15 s, 72 °C for 1 min. Finally, 5 min at 72 °C for full extension. All of the amplified products were sub-cloned into the pEASY-Blunt3 Cloning vector (Transgen, China) and transformed into Trans1-T1 Phage Resistant Chemically Competent Cell for sequencing. All primers are listed in Table S1. 2.4. Sequence alignment and phylogenetic analysis Multiple alignment of CYC2c protein sequences from Chrysanthemum morifolium cv. Hj and other selected species were conducted using ClustalX 2.0 and DNAMAN 7 with default parameters. Then we used the proximity method (Neighbor-Joining method) with the Poisson model and 1000 bootstrap replicates by MEGA7 software to generate the phylogenetic tree and edited by the online tool (https:// itol.embl.de/).

2.6. Determination of medicinal ingredients RFs and DFs from the same inflorescence were collected from the three groups of Hj, and pulverized for determination of the content of the medicinal ingredients: chlorogenic acid, luteoloside and 3,5-dicaffeoylquinic acid; the whole florets (WFs) were collected from three groups of treatments for the determination according to the same method. Each group of 0.25 g powder was sonicated (25 W; 45 kHz) in 25 mL 70% methanol for 40 min, filtered through a microporous membrane (pore diameter = 0.45 mm) (Chinese Pharmacopoeia Committee, 2015) and used for the components determination Under a HPLC system (Agilent 1260 HPLC, USA) in Nanjing Agricultural University. Data statistics and analysis were performed using GraphPad Prism 7 software.

2.5. Quantitative real-time PCR analyses For gene analysis, the RFs and DFs of Hj under three groups of SMR (the DFs was opened 50% and the RFs was opened 70%) were collected to detect the expression level of HjCYC2c and CHS, F3H, FLS and DFR in Hj which are critical in the flavonoid synthesis pathway. We have collected one single inflorescence in each plant of each treatment, so we collected 15 samples, then mixed and grinded in liquid nitrogen, three biological replicates and three technical replicates were randomly selected for each sample. The reverse transcription reaction was carried out with Hiscript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, China) following the manual’s recommendations. The cDNA samples were diluted 5 times with 80 ml deionized water for all gene expression analyses. The qRT-PCR reaction was performed with ChanQ Universal SYBR qPCR Master Mix (Vazyme, China) under ABI StepOne Real-Time PCR (ABI, USA)and the relevant gene-specific primer pairs are listed in

3. Results 3.1. Botanical traits statistics Different SMR have certain effects on Hj’s morphology. With the increase of soil moisture content, the inflorescence diameter became smaller, the number of RFs increased significantly, the number of DFs decreased significantly, the proportion of the number of RFs in the total 3

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Table 1 Effect of soil moisture regimes on inflorescence morphological indexes of Hangju. Morphological indexes

S1

S2

S3

the the the the the the the the the the

4.25 ± 0.37a 7.47 ± 1.15a 90.60 ± 13.41a 165.35 ± 43.79a 1.53 ± 0.14a 36.29 ± 4.12c 2.03 ± 0.20a 0.56 ± 0.06a 1.69 ± 0.19a 0.48 ± 0.06a

4.09 ± 0.38a 7.40 ± 1.02a 107.53 ± 12.42b 147.67 ± 33.84a 1.49 ± 0.11a 42.66 ± 5.31b 1.88 ± 0.26a 0.56 ± 0.07a 1.52 ± 0.20b 0.46 ± 0.06a

3.71 ± 0.18b 7.40 ± 1.08a 108.13 ± 18.77b 116.47 ± 19.74b 1.38 ± 0.09b 48.17 ± 6.49a 1.69 ± 0.15b 0.52 ± 0.04a 1.43 ± 0.18b 0.41 ± 0.06b

diameter of the inflorescence (cm) rounds of RFs (rounds) number of RFs (number) number of DFs (number) diameter of all DF groups(cm) number of RFs/total florets (%) length of the corolla tube of external RF (cm) width of the corolla tube of external RF (cm) length of the corolla tube of innermost RF (cm) width of the corolla tube of innermost RF (cm)

Note: Soil moisture levels were 35%–40% (S1), 65%–70% (S2) and 95%–100% (S3) of the maximum water holding capacity, respectively. Among them, S3 is designed to simulate flooding stress. Data are shown as mean ± SD, n = 15. Different lowercase letters in comparison among different SMR indicate significant difference, P < 0.05.

florets increased, the rounds of RFs was no significant change and the diameter of all DFs group was reduced. Moreover, the size of both innermost RFs and external RFs show a decreasing trend (Table 1). It indicated that excessive SMR in flower-bud differentiation period caused the development of flowers to be blocked, especially for DFs. Then we further speculated that the flower primordia originally may develop into DFs was affected to develop into RFs. Meanwhile, during the experiment stage, it was observed that the flowering of Hj in S3 treatment was delayed by 16 days compared to the S1 treatment, the number of flower heads was also reduced and appeared a lot of incomplete or malformed flowers which were charactered by morphologically deformed, the number of ray floret was significantly reduced and the loss of a part of RFs, respectively (Fig. 1J–L).

with significant difference and F3H was slightly up-regulated, while FLS was down-regulated sharply. In S3 treatment, CHS was down-regulated significantly, while DFR and F3H were almost same between S2 and S3, FLS was slightly down-regulated with no significant difference.The most significant change came from FLS gene, and the trend in response to SMR changes in RFs and DFs were diverged. Therefore, we persume that FLS gene is a strong candidate as a key factor in Hj, Which need a further verification through determination of component as downstream products.

3.4. Determination of medicinal ingredients We performed data analysis of medicinal ingredients to explore the correlation of internal quality of Hj and SMR variation. From the results of WF samples, the content of chlorogenic acid and 3,5-dicaffeoylquinic acid increased with the increase of soil water content, but luteoloside has no significant difference (Fig. 4A–C). The content of chlorogenic acid, luteoloside, and 3,5-dicaffeoylquinic acid in WFs of S3 treatment increased by 48.63%, 20.89% and 47.03%, respectively, with respect to the corresponding items in S1 treatment. Among the three medicinal ingredients, chlorogenic acid (should not less than 0.20%) and luteoloside (should not less than 0.080%) all reached the minimum content of the component in the dried basis specified in the Pharmacopoeia. Only 3,5-dicaffeylquinic acid (should not less than 0.70%) did not reach the corresponding content standard in all three treatments, and we presume that with the changes in soil moisture conditions of Hj in the flower-bud differentiation period, the corresponding changes in the content of 3,5-dicaffeylquinic acid are crucial factors affecting the quality of geo-herbalism and determination in practice. From the determination results of medicinal ingredients in both RFs and DFs, the increasing tendency of the various components in DFs was more obvious than that in RFs, and the maximum value all appeared in the S3 treatment (Fig. 4D–I). The content of chlorogenic acid, luteoloside, and 3,5-dicaffeoylquinic acid of RFs in S3 treatment increased by 37.28%, 4.51% and 27.04%, respectively, compared with the corresponding components of S1 treatment; while the content of chlorogenic acid, luteoloside, and 3,5-dicaffeoylquinic acid of DFs in S3 treatment surged 94.99%、68.38%、85.27% than that corresponding content of S1 treatment. The chlorogenic acid content in RFs of S1, S2 and S3 treatment increased by 42.92%、15.06%、0.62% than that of DFs, respectively; the luteoloside content in RFs of S1, S2 and S3 treatment increased by 31.07%、−6.53%、−18.65% than that of DFs, respectively; the 3,5-dicaffeoylquinic acid content in RFs of S1, S2 and S3 treatment increased by 4.70%、−13.46%、−28.21% than that of DFs, respectively. Furthermore, with the increase of soil moisture content, all of three medicinal ingredients in DFs have a tendency to go ahead with the corresponding components in RFs, and we further speculated that the DFs respond more quickly to the variation of SMR.

3.2. Sequence analysis of the chrysanthemum CYC2c homologs A CYCLOIDEA homolog was isolated from C. morifolium Hj and named as HjCYC2c (GenBank accession No. MK894825). HjCYC2c has a 900 bp opening reading frame, which encodes putative polypeptides of 299 amino acids. Phylogenetic analysis revealed that HjCYC2c belongs to the TCP family, and contains a well-conserved basic helix-loop-helix TCP domain (Fig. 2A). Apparently, all inclusive genes were found to have TCP domains differ in the lengths (Fig. 2B). The Neighbor-Joining method analysis placed all the CYCLOIDEA-like protein genes of Asteraceae into one clade while two Scrophulariaceae plants were sorted with Arabidopsis thaliana of Brassicaceae as a whole. The most clearly supported orthologous for HjCYC2c were CmCYC1 (ALP75557.1) and ClCYC3 (AQQ72783.1). Also, HjCYC2c sisters to CmCYC2 (ALP75558. 1) and CmCYC2c (AMR68927.1), indicating that the CYCLOIDEA genes in Asteraceae shared duplications which were well-conserved and lineage-specific. 3.3. Expression patterns of related genes in Hj Given that the quantitative RT-PCR (qRT-PCR) assay data to detect the expression patterns of HjCYC2c in Hj, we found that the HjCYC2c gene was mainly expressed in RFs, while it was weakly expressed in DFs. Compared with the control group S2 treatment, the expression of HjCYC2c was significantly up-regulated with the increase of SMR, and the response to flooding stress was greater than S1 group (Fig. 3A–B). However, in the DFs sample, both S1 and S3 treatment caused a certain degree of up-regulation of HjCYC2c comparing to the S2 group of normal water conditions. In addition, in the qRT-PCR results of key genes in the flavonoid synthesis pathway, with the change of SMR (S3, S1 treatment compared with S2), restricted to RFs, CHS gene showed a certain degree of downregulation, while the DFR, F3H and FLS genes all showed a certain degree of up-regulation (Fig. 3C–J). However, the trend in DFs was more complicated. In S1 treatment, CHS and DFR were up-regulated 4

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Fig. 2. Multiple alignment and phylogenetic analysis based on CYC-like proteins from Asteraceae and other selected organisms. (A) Complete alignment of deduced amino acid sequences of HjCYC2c and other CYC-like proteins. The identical amino acid residues are shaded in black, similar indicated in pink (identity≥75%), purple (identity≥50%). (B) Phylogenetic analysis of selected CYC-like proteins with the Neighbor-Joining method. The rectangle legends represents the TCP domain, the dot legend represents the relative bootstrap value. The accession numbers of all listed amino acid sequences retrieved from GenBank database are shown following gene names (Given that HjCYC2c is newly identified and named in this study, only the accession numbers of mRNA sequence was listed). Specie abbreviations are listed in Table S2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

4. Discussion

further supported that the variation in HjCYC2c sequence didn’t make the morphological difference. We need do further studies to elucidate if there are any variations in expression patterns of the HjCYC2c.

4.1. High SMR condition relatively promotes the development of RFs Whole plants respond to drought through morphological, physiological, and metabolic modifications occurring in all plant organs (Cellier et al., 1998). From the aspect of morphology, we can see that the most significant phenotype divergence lies in the item for number of ray florets/total florets (Table 1), with the increase of SMR, the whole inflorescence tends to be more compact, and the size is reduced, especially for DFs. Under high SMR condition, it’s apparently that the energy and nutrients flowed into the RFs’ development pathway and promoted the differentiation of RFs, while DFs were relatively inhibited. Distinguished from several times flooding stress in the period of flower-bud differentiation, the prolonged excessive or little SMR has great impact on plant morphology. Flooding regimes of different depths and durations impose selection pressures for various traits in terrestrial wetland plants (Colmer and Voesenek, 2009). In the original cultivation areas of Hj, the continuous rainy season makes the chrysanthemum in an excessive water condition for a long time in the flower-bud differentiation period. Therefore, compared to greenhouse conditions, the stress of low oxygen status of the soil and the scorching weather may bring more impact on plants. So we can explain why as the latitude rises, the annual precipitation decreases, and the disturbance of the rainy weather during the flower bud differentiation period is avoided, the inflorescences in other northern introduced areas are larger on inflorescence morphology, and the diameter of all DF groups is tend to be larger, the number of RFs is increased (Wang et al., 2012). In the Chinese Pharmacopoeia, Hj is listed as one of the five major medicinal chrysanthemums, and ZY is one of the main cultivars of Hj. Given that ZY is weak in resisting the stress such as high temperature and waterlogging, so it is necessary to monitor and regulate the water conditions in actual cultivation.

4.3. Expression analysis indicates the up-regulation for both HjCYC2c and FLS gene The expression patterns of HjCYC2c in DFs and RFs showed the upregulated of this gene with high SMR condition. Combined with the results of botanical traits statistics, high SMR treatment resulted high gene expression and the proportion of RFs/WFs was also increased. In spite of the impaired development for both RFs and DFs, it seems that more RFs represent a tendency to bilateral symmetry for the whole inflorescence. Based on our research experience on flavonoid compounds, then we conducted the expression pattern of some selected genes in biosynthesis pathway of flavonoids which our group have cloned previously, expecting to find out some genes of a noticeable change for follow-up molecular research. The biosynthesis pathway of flavonoids is a complex process. In this study, the expression patterns of F3H and DFR gene in four candidate genes were similar and the expression level were not significant, while the expression levels of CHS gene and FLS gene varied with SMR and the trend were opposite. The CHS gene is located in the upstream of the synthetic pathway, and this results showed that in the case of excessive water, the CHS gene was down-regulated both in RFs and DFs, especially in RFs. With the increase of SMR, the FLS gene showed diametrically opposite changes in RFs and DFs. It is speculated that the degree of response to water stress is different between the RFs and DFs. In this study, the expression of FLS gene in DFs was decreased slightly under S3 treatment, but in RFs was increased greatly. It was estimated that the overall trend in the whole inflorescence showed an increasing trend, namely that the FLS gene showed an increasing trend in the WFs sample under flooding stress. Flavonol synthase (FLS) is a key enzyme in the flavonol branch and flavonols as its downstream products are the most abundant flavonoids in plants (Deng and Lu, 2017). Previous research has been suggested that TCP3 interacts with R2R3-MYB proteins and promotes flavonoid biosynthesis in Arabidopsis thaliana (Li and Zachgo, 2013). Therefore, in view of the up-regulation of FLS and HjCYC2c under the treatment of different SMR, we speculate that there may exist some interactions between the genes regulating for flower symmetry and the biosynthesis pathway of flavonols. On the other hand, there are four key genes have been shown to control dorsoventral asymmetry in Antirrhinum: CYCLOIDEA (CYC), DICHOTOMA (DICH), RADIALIS (RAD), and DIVARICATA (DIV), and this study on Antirrhinum revealed that floral asymmetry involves an interplay between TCP and MYB transcription factors (Corley et al., 2005). Furthermore, it remains to be studied whether some flower symmetry transcription factors encoding MYB proteins interact with proteins regulating for biosynthesis pathway of flavonols. Afterall, accurate genetic control of the different steps of flower development is achieved by a hierarchy of interacting regulatory genes, constituting a gene regulatory network (Kaufmann et al., 2005).

4.2. Duplication and divergence of chrysanthemum CYC2c genes Asteraceae is the largest family of flowering plants, and its inflorescence are highly compressed and solitary (Zhao et al., 2016). Edge effects are often seen, ranging from a mere crowding of the outermost flowers to the formation of additional flower types (Harris, 1999). The flower symmetry biological research species in Asteraceae mainly involved Senecio vulgaris, Helianthus annuus, Gerbera hybrid (Chapman et al., 2012; Tahtiharju et al., 2012; Juntheikki-Palovaara et al., 2014; Garces et al., 2016), and little research on medicinal chrysanthemum. In sunflower, HaCYC2c is necessary for floral zygomorphy, with individuals that are homozygous for a loss-of-function mutation (either due to a premature stop codon or to a reduction/loss of expression of HaCYC2c) exhibiting actinomorphic RFs (Chapman et al., 2012). In Chrysanthemum morifolium, the data in transgenic lines show that overexpression of CmCYC2c leads to elongated ventral ligule length in RFs (Huang et al., 2016). In order to verify the problem whether the morphological difference in different cultivar’s inflorescence of Chrysanthemum morifolium is due to gene sequence or not, we conducted multiple sequence alignment. In this study, we found that the sequence variation between HjCYC2c and CmCYC2c (ornamental Chrysanthemum) was few, indicating that CYC2c gene shared well-conserved sequence during different cultivars of Chrysanthemum morifolium. Furthermore, the results of little divergence in the sequence are

4.4. The accumulation of active ingredients increased under S3 treatment With water status of too little or too much is a major factor resulting in plant stress and adversely affect many aspects of plant physiology, especially for suppression of root metabolism (Mielke and Schaffer, 6

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Fig. 3. The relative expression levels of the HjCYC2c and some key genes in flavonoid synthesis pathway. Soil moisture levels were 35%–40% (S1), 65%–70% (S2) and 95%–100% (S3) of the maximum water holding capacity, respectively. (A–J) Expression patterns of HjCYC2c, CHS, F3H, FLS and DFR in RFs and DFs samples under different treatments. The values were normalized to the β-Actin, with error bars depicting the standard deviation of three biological replicates; n.s., no significance; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, Student t-test.

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Fig. 4. The determination data of three medicinal ingredients (chlorogenic acid, luteoloside, and 3,5-dicaffeoylquinic acid) in different samples. Soil moisture levels were 35%–40% (S1), 65%–70% (S2) and 95%–100% (S3) of the maximum water holding capacity, respectively. (A–C) The determination data of three medicinal ingredients in whole flower (WFs). (D–F) The determination data of three medicinal ingredients in ray florets (RFs). (G–I) The determination data of three medicinal ingredients in disc florets (DFs). Error bars show SD; n.s., no significance; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, Student t test, n = 3 biological replicates.

hand, being different from the crops aiming for yields, the cultivation of medicinal plants also lay emphasis on stability of property of a medicine. Moreover, a study on carrot provide ideas for us that when plants are treated with water stress alone or when additional water stress is applied on wounded tissue, it can induce the accumulation of specific primary or secondary metabolites (Becerra-Moreno et al., 2015). Obviously, the idea is deserved more attention for research: the geo-authentic habitat state of medicinal plants directly affects the content of active ingredients and medicinal stability. Therefore, monitoring and regulating the water conditions in Hj’s actual cultivation are the effective approaches for better stability of this medicinal material.

2010; Yamauchi et al., 2018). The effects of SMR on plant secondary metabolites varied by diverse species and pathways. The study about Artemisia annua L. has revealed that the secondary metabolites including artemisinin, arteannuin B, artemisinic acid ＋dihydroartemisinic acid, and essential oil content were positively controlled by the developmental program however negatively modulated by water stress (Yadav et al., 2014). However, in a study about Arabidopsis thaliana (L.), they found that drought stress and water-logging led to increased aliphatic glucosinolate and flavonoid levels (Mewis et al., 2012). In this study, the higher SMR condition (S3) have helped generate more secondary metabolites, consisting with our previous studies (Wang et al., 2014). Furthermore, the flooding stress during flower-bud differentiation can significantly enhance the accumulation of active ingredients (Zou et al., 2018). It is important to understand the metabolic pathways of secondary metabolites, to predict which compounds are likely to increase or decrease, and to comprehend the complex interaction of environmental influences on plants (Prinsloo and Nogemane, 2018). Distinguished from the requirements of morphology and breeding for ornamental flowers, medicinal plants are relatively conservative in the evolution process of thousands of years due to the particularity for their medicinal value, which gradually formed the authentic quality. On the other

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author contributions QS Guo, T Wang and WY Zhang designed the research project; WY Zhang, DH Lu and J Liu performed the research; WY Zhang analyzed data and wrote the paper. WY Zhang, QJ Zou and F Yang modified the 8

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paper. All the authors read and approved the manuscript.

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