Abstract

Simple Summary

   This study revealed the internal regulation mechanism of Hibiscus
   mutabilis discoloration three times a day through joint Transcriptome
   and metabonomics, which promoted the molecular breeding of ornamental
   plants.

Abstract

   In this study, we used combined transcriptomics and metabolomics to
   analyze the H. mutabilis cultivar’s genetic and physiological
   mechanisms during three flower color transition periods (from white to
   pink, then from pink to red) within the span of one day. As a result,
   186 genes were found to be significantly increased with the deepening
   of the H. mutabilis flower color; these genes were mainly involved in
   the expression of peroxidase 30, zinc finger protein, phosphate
   transporter PHO1, etc. In contrast, 298 genes were significantly
   downregulated with the deepening of H. mutabilis flower color,
   including those involved in the expression of probable
   O-methyltransferase 3, copper binding protein 9, and heat stress
   transcription factor A-6b. Some genes showed differential expression
   strategies as the flower color gradually darkened. We further detected
   19 metabolites that gradually increased with the deepening of the H.
   mutabilis flower color, including L-isoleucine, palmitic acid,
   L-methionine, and (+)-7-isonitrobenzene. The content of the metabolite
   hexadecanedioate decreased with the deepening of the H. mutabilis
   flower color. Combined transcriptomics and metabolomics revealed that
   the metabolic pathways, including those related to anthocyanin
   biosynthesis, cysteine and methionine metabolism, and sulfur
   metabolism, appear to be closely related to H. mutabilis flower color
   transition. This study served as the first report on the genetic and
   physiological mechanisms of short-term H. mutabilis flower color
   transition and will promote the molecular breeding of ornamental
   cultivars of H. mutabilis.

   Keywords: flower color transition, mechanism, breeding, gardening,
   metabolic pathway, gene regulation

1. Introduction

   Hibiscus mutabilis is an important ornamental plant from the Malvaceae
   family that originated in China and has been cultivated in China for
   more than 2000 years [[34]1]. As an ornamental plant, H. mutabilis has
   spread to Japan, Korea, and other countries in Asia. Species of the
   Malvaceae family are considered to have great economic and ornamental
   value, and include species such as Gossypium hirsutum and Hibiscus
   syriacus (Rose of Sharon). The former is the main global source of
   natural textile fiber production [[35]2], and the latter is the
   national flower of Korea, and has attractive white, pink, red,
   lavender, or purple flowers and a long bloom period [[36]3]. H.
   mutabilis is also studied worldwide because of its attractive flowers,
   which have a long bloom period, generally 3–4 months, although the
   persistence of individual flowers lasts for fewer than 48 h. Unlike
   some other ornamental plants, such as chrysanthemums [[37]4], Narcissus
   pseudonarcissus [[38]5], and Brassica napus [[39]6], different flower
   colors exist among different cultivars. Some H. mutabilis cultivars can
   show rapid flower color change on the same day. The most famous
   cultivar is ‘Drunk Girl’, also known as ‘Zui Furong’, which can display
   three different colors over the course of one day, from white in the
   morning to pink at noon and dark red in the evening. This obvious color
   change has attracted a large number of visitors from around the world.
   H. mutabilis bears important cultural and historical significance in
   the city of Chengdu, and is regarded as the city flower.

   In addition to being an ornamental plant, H. mutabilis is also used as
   a source of traditional medicine in China; its functions of
   detoxification, detumescence, and pain relief have led to it being used
   for a long time to treat ulcers, swelling, herpes zoster, burns, and
   bruises [[40]7,[41]8,[42]9,[43]10]. Modern medical research has shown
   that the main active ingredients in H. mutabilis include flavonoid
   glycosides, ferulic acid, lectin, rutin, and isoquercetin, which have
   antioxidant, anti-insect, anti-viral, and anti-inflammatory effects
   [[44]11,[45]12,[46]13,[47]14]. The flowers of H. mutabilis have also
   been shown to contain a variety of bioactive substances, such as
   flavonoids, rutin, and quercetin [[48]13]. Despite being an important
   ornamental and medicinal plant, the mechanism of flower color change in
   H. mutabilis is still unknown. The gene regulation mechanisms of flower
   color change and of the change in metabolites need to be further
   analyzed.

   In this study, we collected H. mutabilis flower samples during three
   color transition periods and then used transcriptomics and metabolomics
   to reveal the gene regulation and metabolic mechanism of the H.
   mutabilis color transition process. The research results revealed the
   genetic and physiological mechanisms of H. mutabilis color transition
   during three time periods in a day, which will provide guidance for the
   molecular breeding of ornamental plants and the development or
   utilization of natural active ingredients in H. mutabilis.

2. Materials and Methods

2.1. Sample Collection

   We collected different colors of H. mutabilis flowers from one H.
   mutabilis strain in the Chengdu Botanical Garden in Sichuan, China.
   During one day of the H. mutabilis bloom period, we collected 6 white
   flowers at 8:00, 6 pink flowers at 13:00, and 6 red flowers at 17:00,
   all from the same plant ([49]Figure 1). The collected flowers were
   stored on dry ice and then returned to the laboratory for subsequent
   RNA and metabolite extraction. White H. mutabilis flowers were assigned
   the code ZFRW, pink ones the code ZFRP, and red ones the code ZFRR in
   subsequent analyses.

Figure 1.

   [50]Figure 1
   [51]Open in a new tab

   H. mutabilis flowers of different colors collected from the same plant
   at different times of the day. ZFRW, white H. mutabilis flowers; ZFRP,
   pink H. mutabilis flowers; ZFRR, red H. mutabilis flowers.

2.2. RNA Preparation and Library Construction

   The total RNA of collected flowers was extracted using a plant RNA Kit
   (Omega Bio-Tek, Norcross, GA, USA). The extracted RNA was monitored on
   1% agarose gels. The RNA concentration was measured using a Qubit^® RNA
   Assay Kit and a Qubit^® 2.0 Fluorometer (Life Technologies, Carlsbad,
   CA, USA). A total amount of 1.5 µg of RNA per sample was used as the
   input material for RNA sample preparation. We constructed RNA
   sequencing libraries using the NEBNext^® Ultra™ RNA Library Prep Kit
   for Illumina^® (NEB, Ipswich, MA, USA) according to the manufacturer’s
   instructions. The quality of the obtained RNA sequencing libraries was
   assessed with the Agilent Bioanalyzer 2100 system (Agilent
   Technologies, Santa Clara, CA, USA). The libraries were then sequenced
   on an Illumina HiSeq platform, and paired-end reads were generated.

2.3. Quality Control and Transcriptome Assembly

   The obtained paired-end reads were first processed through quality
   control steps to generate clean reads. Briefly, raw reads with low
   quality and containing adapter sequences or poly-N stretches were
   removed using Perl scripts. We further calculated the Q20, Q30, and GC
   contents and sequence duplication level of the clean data. All
   downstream analyses were based on clean data with high quality. We used
   Trinity to assemble the transcriptome with min_kmer_cov set to 2 by
   default and all other parameters set to default [[52]15]. The assembled
   transcripts were functionally annotated according to seven databases,
   including Nr (NCBI nonredundant protein sequences) [[53]16], Nt (NCBI
   nonredundant nucleotide sequences) [[54]16], Pfam (Protein family)
   [[55]17], KOG/COG (Clusters of Orthologous Groups of proteins)
   [[56]18], Swiss-Prot (a manually annotated and reviewed protein
   sequence database) [[57]19], KO (KEGG Ortholog database) [[58]20], and
   GO (Gene Ontology) [[59]21].

2.4. Differentially Expressed Gene Analysis

   Clean data were first mapped back onto the assembled transcriptome.
   Read counts for each gene were obtained from the mapping results. We
   calculated the gene expression level for each sample using RSEM
   [[60]22]. We performed differential expression analysis using the DESeq
   R package [[61]23]. The resulting p values were adjusted using
   Benjamini and Hochberg’s approach for controlling the false discovery
   rate. Genes with an adjusted p-value of <0.05 were considered
   differentially expressed.

   Gene ontology (GO) enrichment analysis of the differentially expressed
   genes (DEGs) was conducted with the GOseq R package using the default
   Wallenius noncentral hypergeometric distribution [[62]24]. The
   statistical enrichment of differentially expressed genes in KEGG (Kyoto
   Encyclopedia of Genes and Genomes) pathways was tested using KOBAS 3.0
   software [[63]25].

2.5. Extraction of Metabolites

   Approximately 200 mg of a petal sample was added to a 2 mL Eppendorf
   tube, and 0.6 mL of 2-chlorophenylalanine (4 ppm) methanol (−20 °C) was
   then added and vortexed for 30 s. The mixture was ground using a tissue
   grinder (DHTISSUE-32, LAWSON, Ningbo, China) for 90 s at 55 Hz. The
   ground plant tissue mixture was sonicated at room temperature for 15
   min. We then centrifuged the mixture at 12,000 rpm for 10 min and took
   200 μL supernatant by using 0.22 μM filter membrane filtration, and the
   collected filter solution was used for subsequent detection. All of the
   samples were randomly injected into the apparatus to avoid system
   errors. We also conducted quality control (QC) to ensure the stability
   of the system.

2.6. Metabolome Detection

   We used a Thermo Ultimate 3000 system and an ACQUITY UPLC^® HSS T3 (150
   × 2.1 mm, 1.8 µm, Waters, Milford, MA, USA) column for the
   chromatographic detection of the extracted metabolites. The temperature
   of the automatic sampler was set to 8 °C, the flow rate was 0.25
   mL/min, the column temperature was 40 °C, and the sample was injected
   at 2 μL for gradient elution. The ESI-MSn experiments were executed on
   a Thermo Q Exactive Focus mass spectrometer with spray voltages of 3.5
   kV and −2.5 kV in positive and negative modes, respectively. The
   detection conditions of metabolites were set according to those in
   previously published papers.

2.7. Metabolite Data Analysis

   We used Waters Masslynx 4.1 software to extract metabolite peaks from
   the LC–MS/MS data. Proteowizard software (v3.0.8789) was then used to
   turn the raw MS files into the mzXML format, which were sequentially
   processed using XCMS software for chromatographic matching, metabolic
   feature detection, and metabolite peak alignment. The metabolite peak
   areas were normalized by the sum of the areas and subjected to
   statistical analyses.

   Soft Independent Modeling of Class Analogy (SIMCA)-P (version 11.0,
   Umetrics AB, Umeå, Sweden) was used to conduct multivariate analyses.
   All variables were UV (Unit Variance) scaled before partial least
   squares-discriminant analysis (PLS-DA). We used a loading plot to show
   how different variables contributed to the samples. Based on the
   loading plot in PLS-DA, notably different metabolites were identified.
   Afterward, t-tests were performed independently to determine
   significant metabolites (p ≤ 0.05) between the groups. An analysis of
   pathway topology on Metaboanalyst [[64]26] and the KEGG metabolic
   database [[65]20] was conducted to map metabolic pathways.

   The Pearson correlation coefficient was used to analyze the correlation
   between metabolite expression and transcriptional expression. A
   metabolic pathway was drawn and visualized using Cytoscape 3.02
   [[66]27].

3. Results

3.1. Overview of the Transcriptome Data

   To study the gene regulation mechanism of H. mutabilis flower color
   change over the course of one day, we analyzed the gene expression
   profiles of H. mutabilis flowers from three color transition periods by
   transcriptome sequencing. On average, 6.52 Gbp of raw RNA data was
   obtained per sample, with a Q20 and Q30 of 97.78% and 93.84%,
   respectively ([67]Table S1). We conducted quality control steps to
   remove reads with low quality from the raw RNA reads, and approximately
   11.20% of the raw reads were discarded. An average of 38,570,298 clean
   reads per sample were used for subsequent analysis. The obtained clean
   reads were used to assemble H. mutabilis transcripts and unigenes. A
   total of 352,980 transcripts and 116,127 unigenes were obtained, with
   total lengths of 352.11 Mbp and 95.91 Mbp, respectively ([68]Table S2).
   The GC contents of the obtained transcripts and unigenes were 41.32%
   and 40.64%, respectively. We further annotated the H. mutabilis
   unigenes against the seven databases ([69]Table S3); 62.75% of the
   unigenes could be annotated in the NR database, 58.44% could be
   annotated in the eggNOG database, and a total of 64.46% could be
   annotated in at least one of the seven databases.

3.2. Gene Expression Analysis

   We analyzed the expression profiles of H. mutabilis in different color
   transition periods and analyzed the correlation between the expression
   profiles of different samples ([70]Figure 2). The results showed that
   H. mutabilis samples from the same color transition period had a
   greater correlation, while the expression profiles from different color
   transition periods showed significant differences. The expression
   profiles of white and pink H. mutabilis were more similar to each other
   than they were to those of red samples. The analysis of differentially
   expressed genes showed that 16,451 differentially expressed genes were
   found between white and pink flowers, including 8757 upregulated genes
   and 7694 downregulated genes in pink flowers relative to white flowers
   ([71]Figure 3). There were 8484 differentially expressed genes in pink
   and red H. mutabilis flowers, including 4206 upregulated genes and 4278
   downregulated genes in red H. mutabilis flowers relative to those in
   pink H. mutabilis flowers. We detected 13,323 specific differentially
   expressed genes between the white and pink H. mutabilis flower samples
   and 5356 specific differentially expressed genes between the pink and
   red H. mutabilis flower samples ([72]Figure 4). A total of 3128 common
   differentially expressed genes were detected between the white and pink
   H. mutabilis samples and between the pink and red H. mutabilis flower
   samples.

Figure 2.

   [73]Figure 2
   [74]Open in a new tab

   Correlation and cluster analysis of gene expression profiles for H.
   mutabilis flower samples of different colors.

Figure 3.

   [75]Figure 3
   [76]Open in a new tab

   Volcano map of differentially expressed genes between white and pink
   flowers (a) and between pink and red flowers (b). ZFRW, white H.
   mutabilis flowers; ZFRP, pink H. mutabilis flowers; ZFRR, red H.
   mutabilis flowers.

Figure 4.

   [77]Figure 4
   [78]Open in a new tab

   Shared and specific differentially expressed genes between different
   samples. ZFRW, white H. mutabilis flowers; ZFRP, pink H. mutabilis
   flowers; ZFRR, red H. mutabilis flowers.

3.3. Genes Upregulated with Darker Flower Color

   Statistical analysis showed that the expression of 186 genes
   significantly increased with the deepening of H. mutabilis flower
   color. These mainly included genes such as those involved in the
   expression of peroxidase 30, zinc finger protein, phosphate transporter
   PHO1, cytochrome P450 82c4, basic leucine zipper 1, peroxidase 54,
   oligopeptide transporter 4, and cytochrome c1 ([79]Table S4).

   In addition, there were 4389 genes that were upregulated in the process
   of H. mutabilis flowers transitioning from white to pink, but there was
   no significant change in the expression level during the transition
   from pink to red. The genes upregulated in the transition from white to
   pink included those involved in the expression of zinc finger
   homeodomain protein, fumarylacetase, malate dehydrogenase,
   methylenetetrahydrofolate reductase 2, transcription reporter myb5,
   ATP-dependent zinc metalloproteinase FtsH, probable acyl activating
   enzyme 2, probable transcription factor kan2, high-affinity nitrate
   transporter 2.5, calcium-dependent protein kinase 9, and flavanone
   3-dioxygenase 1.

3.4. Genes Downregulated with Darker Flower Color

   There were 298 genes that were significantly downregulated with the
   deepening of H. mutabilis flower color, such as genes that are mainly
   involved in the expression of probable O-methyltransferase 3, copper
   binding protein 9, fatty acid biosynthesis 1 isoform 1, heat stress
   transcription factor A-6b, heat stress transcription factor C-1, auxin
   response factor 7, molybdenum cofactor sulfurase, glutamate
   formimidoyltransferase, and short-chain dehydrogenase reductase 2a
   ([80]Table S4).

   We detected 3388 genes that were first upregulated during the H.
   mutabilis flower color transition from white to pink and that then
   showed no significant change during the transition from pink to red.
   The upregulated genes included those involved in the expression of
   auxin response factor 11, metal tolerance protein 4, anthranilate
   synthase beta subunit 2, probable methyltransferase PMT28, probable
   inactive receptor kinase At4g23740, mini zinc finger protein 2, signal
   peptide peptidase-like 2, and ubiquinone biosynthesis COQ4.

3.5. Genes Differentially Expressed with Darker Flower Color

   The expression levels of 114 genes increased first and then decreased
   as the flowers of H. mutabilis changed from white to pink and then to
   red. These differentially expressed genes included those involved in
   the expression of chalcone synthase 2, protochlorophyllide reductase,
   oxygen-evolving enhancer protein 1, photosystem I reaction center
   subunit XI, UDP-glycosyltransferase 74B1, homeobox-leucine zipper
   protein ATHB-6, and probable metal-nicotianamine transporter YSL5
   ([81]Table S4).

   In addition, a total of 9 genes significantly decreased and then
   increased as the flowers of H. mutabilis changed from white to pink and
   then to red. These genes included those involved in the expression of
   meiotic nuclear division protein 1, arginine decarboxylase, WD
   repeat-containing 62, organic cation/carnitine transporter 3, plant
   cysteine oxidase 1, aquaporin PIP2-1, and haloacid dehalogenase-like
   hydrolase superfamily protein.

   We also detected 658 genes that showed no significant change during the
   H. mutabilis flower color transition from white to pink but that were
   then significantly downregulated during the transition from pink to
   red. These genes included those involved in the expression of
   photosystem I reaction center subunit V, ribulose bisphosphate
   carboxylase small chain, serine hydroxymethyltransferase, probable
   sulfate transporter 3.5, protochlorophyllide reductase, photosystem I
   subunit O, homeobox-leucine zipper protein HDG1, and serine protease
   SPPA.

3.6. GO Functional Enrichment Analysis

   We further conducted a GO enrichment analysis of the DEGs between the
   white and pink H. mutabilis flower samples ([82]Figure 5a). The results
   showed that molecular functions related to oxidoreductase activity,
   catalytic activity, and transporter activity; cellular components
   related to the extracellular region; and biological processes related
   to the oxidation—reduction process, aromatic amino acid family
   metabolic process, and inorganic anion transport were all significantly
   enriched in the pink H. mutabilis flower samples compared with those in
   the white flower samples. In addition, the results showed that cellular
   components related to the membrane and its intrinsic and integral
   components, and that biological processes related to responses to
   auxin, cell wall organization, and external encapsulating structure
   organization, were significantly decreased in the pink H. mutabilis
   flower samples compared with those in the white flower samples.

Figure 5.

   [83]Figure 5
   [84]Open in a new tab

   Gene ontology (GO) enrichment analysis of the differentially expressed
   genes (DEGs) between white and pink flowers (a) and between pink and
   red flowers (b).

   In the process of H. mutabilis changing from pink to red, some cellular
   components and molecular functions were significantly enhanced. This
   included cellular components, such as those related to the ribosomal
   subunit, the ribosome, and the cytosolic ribosome, and molecular
   functions, such as those related to the structural constituents of the
   ribosome and structural molecule activity ([85]Figure 5b). However,
   molecular functions related to oxidoreductase activity, tetrapyrrole
   binding, and cofactor binding were significantly reduced. Biological
   processes related to the auxin-activated signaling pathway, the
   cellular response to auxin stimulus, and the response to auxin were
   also significantly reduced, as were cellular component DEGs related to
   the thylakoid, photosynthetic membrane, and chloroplasts.

3.7. KEGG Pathway Enrichment Analysis

   KEGG pathway enrichment analysis for the H. mutabilis flower samples
   showed that, compared with white flower samples, the pink flower
   samples were significantly enriched in pathways related to
   phenylpropanoid biosynthesis; flavonoid biosynthesis; phenylalanine
   metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis;
   glycerolipid metabolism; starch and sucrose metabolism; and tryptophan
   metabolism ([86]Figure 6a). Compared with the white flower samples, the
   pink flower samples were significantly decreased in pathways related to
   plant hormone signal transduction, steroid biosynthesis, fatty acid
   elongation, and cutin, suberine, and wax biosynthesis.

Figure 6.

   [87]Figure 6
   [88]Open in a new tab

   KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment of
   differentially expressed genes between white and pink flowers (a) and
   between pink and red flowers (b). ZFRW, white H. mutabilis flowers;
   ZFRP, pink H. mutabilis flowers; ZFRR, red H. mutabilis flowers.

   When the H. mutabilis flowers changed from pink to red, the pathways
   were significantly enhanced for ribosome, pentose, and glucuronate
   interconversions, as were the pathways for oxidative phosphorylation.
   The KEGG pathways that were significantly decreased included those
   related to plant hormone signal transduction; photosynthesis—antenna
   proteins; photosynthesis; tyrosine metabolism; isoquinoline alkaloid
   biosynthesis; flavonoid biosynthesis; tropane, piperidine, and pyridine
   alkaloid biosynthesis; the pentose phosphate pathway; and phenylalanine
   metabolism ([89]Figure 6b).

3.8. Metabolomic Profiles of H. mutabilis Flowers

   We used the metabolome method to analyze the metabolic profiles of the
   flowers of H. mutabilis from the three color transition periods, and a
   total of 273 metabolites were detected. The OPLS-DA clearly
   distinguished the different flower colors of H. mutabilis samples
   according to their metabolic profiles, showing that there were
   significant differences in the metabolites of the three different H.
   mutabilis color samples ([90]Figure 7).

Figure 7.

   [91]Figure 7
   [92]Open in a new tab

   Partial least squares-discriminant analysis (PLS-DA) of metabolic
   profiles of different samples. ZFRW, white H. mutabilis flowers; ZFRP,
   pink H. mutabilis flowers; ZFRR, red H. mutabilis flowers.

3.9. Differentially Expressed Metabolites

   The statistical analysis revealed that the expression of 19 metabolites
   gradually increased with the deepening of the H. mutabilis flower
   color. These metabolites included L-isoleucine, palmitic acid,
   L-methionine, (+)-7-isocyanmonic acid, acetylcholine,
   3,4-dihydroxybenzeneacetic acid, 2-keto-6-acetamidocaproate, and
   3-O-methylgallate. The content of one metabolite gradually decreased
   with the deepening of the H. mutabilis flower color, and that
   metabolite was hexadecanedioate ([93]Figure 8).

Figure 8.

   [94]Figure 8
   [95]Open in a new tab

   Cluster heatmap analysis of differentially expressed metabolites among
   different samples. ZFRW, white H. mutabilis flowers; ZFRP, pink H.
   mutabilis flowers; ZFRR, red H. mutabilis flowers.

   A total of 20 metabolites in the H. mutabilis flowers showed no
   significant change in expression during the transition from white to
   pink. However, the expression of these 20 metabolites increased
   significantly during the transition from pink to red. These metabolites
   included nalbuphine, trans-trans-muconic acid, 2-isopropylmalic acid,
   guanidinosuccinic acid, D-glucuronic acid, itaconic acid, and stearic
   acid. In addition, 20 metabolites in H. mutabilis showed no significant
   change in expression during the transition from white to pink, while
   the expression decreased significantly during the transition from pink
   to red. These 20 metabolites included guanidoacetic acid, pyruvic acid,
   exemestane, 2-iminobutanoate, phosphoglycolic acid, palmitoleic acid,
   allantoic acid, 2-keto-glutaramic acid, and 3-dehydroshikimate.

   A total of 122 H. mutabilis flower metabolites only increased during
   the transition from white to pink ([96]Figure 9), with no significant
   change in the expression of these metabolites during the transition
   from pink to red. These metabolites included 6-hydroxynicotinate,
   lithocholic acid, nicotinic acid, uric acid, pantothenic acid,
   4-hydroxycinnamic acid, gentisic acid, and sinapic acid. The expression
   levels of four H. mutabilis flower metabolites were significantly
   decreased during the change from white to pink, while the expression
   levels of these metabolites did not change significantly during the
   change from pink to red. These four metabolites were guanosine,
   ketoleucine, 4,5-dihydroorotic acid, and picolinic acid. In addition,
   the content of six metabolites increased first and then decreased as
   the H. mutabilis flower color became darker. These metabolites were
   hydroxykynurenine, methyl beta-D-galactoside, cyclic AMP,
   gamma-aminobutyric acid, 13-L-hydroperoxylinoleic acid, and
   pyrrole-2-carboxylic acid.

Figure 9.

   [97]Figure 9
   [98]Open in a new tab

   Metabolites increased as H. mutabilis flowers transitioned from white
   to pink, but without a significant change in expression as the flowers
   changed from pink to red. ZFRW, white H. mutabilis flowers; ZFRP, pink
   H. mutabilis flowers; ZFRR, red H. mutabilis flowers.

3.10. Transcriptome and Metabolome Association Analysis

   An association analysis based on the KEGG metabolic pathway analysis
   showed that anthocyanin biosynthesis, cysteine and methionine
   metabolism, C5-branched dibasic acid metabolism, glutathione
   metabolism, and beta-alanine metabolism played key regulatory roles in
   the H. mutabilis flower transition from white to pink ([99]Table S5).
   As the H. mutabilis flowers changed from pink to red, sulfur
   metabolism, taurine and hypotaurine metabolism, purine metabolism,
   cyanoamino acid metabolism, glycine, serine, and threonine metabolism,
   vitamin B6 metabolism, amino sugar and nucleotide sugar metabolism, and
   fatty acid metabolism played key regulatory roles.

4. Discussion

   As the city flower of Chengdu, H. mutabilis has important historical,
   cultural, and ornamental value [[100]1]. H. mutabilis is popular with
   tourists because of its variety and ability to change colors over the
   course of one day. The most famous variety is H. mutabilis, the flowers
   of which can transition from white to pink and then red in one day. In
   addition, H. mutabilis is also regarded as an important source of
   Chinese medicine and some natural active substances
   [[101]28,[102]29,[103]30]. The flowers of many plants, such as
   chrysanthemum and monkeyflower [[104]31], can show different colors
   within or between species, and their color changes are considered to be
   closely related to the anthocyanin content or environmental responses
   [[105]32,[106]33,[107]34,[108]35]. However, few plants can change the
   color of their flowers on such a short time scale. To date, the
   physiological and genetic mechanisms of flower color transition over a
   short time period have not been analyzed [[109]36,[110]37]. With the
   development of sequencing and material identification technology,
   scholars can identify the genetic mechanisms of plant flower color
   transition through multiomics [[111]38,[112]39,[113]40,[114]41]. This
   study serves as the first study to analyze the genetic and
   physiological mechanisms of short-term flower color change in H.
   mutabilis by combining transcriptomics and metabolomics, providing an
   important reference for the molecular breeding of ornamental cultivars
   of H. mutabilis.

   The transcriptional analysis in this study revealed that 186 genes
   significantly increased with the deepening of H. mutabilis flower
   color, including those related to the expression of peroxidase 30, zinc
   finger protein, phosphate transporter PHO1, cytochrome P450 82c4, basic
   leucine zipper 1, peroxidase 54, oligopeptide transporter 4, and
   cytochrome c1 [[115]42,[116]43]. Zinc finger proteins have been found
   to play an important role in flower development in various plants
   [[117]44]. Cytochrome P450 enzymes have been found to play an important
   role in the biosynthesis of flavonoids and anthocyanins, which are
   major pigments found in flowers [[118]45,[119]46]. In addition, it has
   been discovered that basic leucine zipper (bZIP) is a key regulator of
   the abscisic acid (ABA) signaling pathway controlling seed dormancy,
   germination, plant growth, and flowering [[120]47,[121]48].
   Furthermore, in this study, 298 genes were significantly downregulated
   with the deepening of H. mutabilis flower color, which mainly included
   genes such as those related to the expression of probable
   O-methyltransferase 3, copper binding protein 9, fatty acid
   biosynthesis 1 isoform 1, heat stress transcription factor A-6b, heat
   stress transcription factor C-1, auxin response factor 7, molybdenum
   cofactor sulfurase, glutamate formimidoyltransferase, and short-chain
   dehydrogenase reductase 2a. In flower pigmentation, O-methyltransferase
   (OMT) plays an important role in the modification of anthocyanins
   [[122]49,[123]50]. Auxin response factors have been reported to play an
   important regulatory role in flower development [[124]51,[125]52]. In
   addition, H. mutabilis has developed a variety of gene regulation modes
   for the short-term flower color change process. Different genes show
   variable expression patterns at different color transition stages. In
   general, thousands of genes are involved in the short-term flower color
   changes of H. mutabilis, and some of them play key regulatory roles in
   flower development and pigment regulation.

   Metabolite analysis showed that there were significant differences in
   metabolite profiles during different H. mutabilis flower color
   transition stages, and the expression of 19 metabolites increased with
   the deepening of the flower color. These metabolites included
   L-isoleucine, palmitic acid, L-methionine, (+)-7-isocyanmonic acid,
   acetylcholine, 3,4-dihydroxybenzeneacetic acid,
   2-keto-6-acetamidocaproate, and 3-O-methylgallate. One metabolite
   gradually decreased with the deepening of the H. mutabilis flower
   color, and that was hexadecanedioate. Interestingly, we did not detect
   the enrichment of common plant pigments, such as flavonoids and
   anthocyanins, during the flower color changes of H. mutabilis
   [[126]53,[127]54,[128]55], which showed that the change in H. mutabilis
   flower color could be the result of the interaction of various
   metabolites [[129]39,[130]56,[131]57]. We also detected metabolites
   specifically enriched at different flower color transition stages;
   these metabolites included 2-iminobutanoate, 4-hydroxycinnamic acid,
   gamma-aminobutyric acid, quercitrin, arachidonic acid,
   3-indoleacrylate, palmitic acid, aspartame, 2-oxoarginine, and methyl
   jasmonate. These natural compounds identified in the H. mutabilis
   flowers have important value in plant growth regulation, the food
   industry, and medicine, and warrant further research and application
   [[132]11,[133]13,[134]58].

   Based on the combined transcriptome and metabolome, we found that the
   metabolic pathways, including those related to anthocyanin
   biosynthesis; cysteine and methionine metabolism; C5-branched dibasic
   acid metabolism; glutathione metabolism; and beta-alanine metabolism
   were closely related to the H. mutabilis color change from white to
   pink. Polyphenol compounds, such as anthocyanins, produce the different
   hues of pink, red, purple, and blue in flowers, vegetables, and fruits
   [[135]59,[136]60,[137]61,[138]62,[139]63,[140]64,[141]65]. The
   enrichment of the anthocyanin biosynthesis pathway may play a key role
   in regulating the color transition of H. mutabilis from white to pink
   [[142]34,[143]66]. In the process of the H. mutabilis flowers changing
   from pink to red, sulfur metabolism, taurine and hypotaurine
   metabolism, purine metabolism, cyanoamino acid metabolism, glycine,
   serine, and threonine metabolism, vitamin B6 metabolism, amino sugar
   and nucleotide sugar metabolism, and fatty acid metabolism played key
   regulatory roles. In general, in the H. mutabilis short-term flower
   color change regulation, multiple regulatory genes, and different
   expression patterns of key genes were involved, resulting in the
   diverse expression of flower metabolites at different color transition
   stages, which jointly promoted the formation and regulation mechanism
   of H. mutabilis flower color. This study served as the first report on
   the regulation and physiological mechanism of short-term flower color
   change in H. mutabilis, and will promote the molecular breeding of
   ornamental cultivars of H. mutabilis.

5. Conclusions

   In this study, a combination of transcriptomics and metabolomics was
   employed to investigate the genetic and physiological mechanisms of the
   H. mutabilis cultivar’s flower color transition over the course of one
   day, from white to pink, then from pink to red. The results revealed
   186 genes that were significantly upregulated and 298 genes that were
   significantly downregulated with the deepening of the flower color.
   These genes were mainly involved in the expression of peroxidase 30,
   zinc finger protein, phosphate transporter PHO1, probable
   O-methyltransferase 3, copper binding protein 9, and heat stress
   transcription factor A-6b. Additionally, 19 metabolites were found to
   increase with deepening flower color, such as L-isoleucine, palmitic
   acid, L-methionine, and (+)-7-isonitrobenzene, while the content of
   hexadecanedioate decreased. The metabolic pathways related to
   anthocyanin biosynthesis, cysteine and methionine metabolism, and
   sulfur metabolism were found to be closely associated with the flower
   color transition. This research serves as the first report on the
   genetic and physiological mechanisms of short-term H. mutabilis flower
   color transition, and could be beneficial in the molecular breeding of
   ornamental cultivars of H. mutabilis.

Supplementary Materials

   The following supporting information can be downloaded at:
   [144]https://www.mdpi.com/article/10.3390/biology12081115/s1, Table S1:
   Transcriptome sequencing and quality control data statistics; Table S2:
   Transcriptome data assembly analysis; Table S3: Unigene annotation
   against the seven databases; Table S4: Expression analysis of different
   genes in different color of ‘Zui Furong’; Table S5: KEGG (Kyoto
   Encyclopedia of Genes and Genomes) pathway enrichment analysis based on
   integrated transcriptomics and metabolomics.
   [145]Click here for additional data file.^ (2MB, zip)

Author Contributions

   Conceived and designed experiments: Q.L., X.L. and Z.Z. Analyzed the
   data: X.Z., X.S. and J.M. Wrote and reviewed the paper: Q.L. All
   authors have read and agreed to the published version of the
   manuscript.

Institutional Review Board Statement

   Not applicable.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   All data generated or analyzed during this study are included in this
   published article (and its [146]Supplementary Materials).

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This study is supported by the special industrial fund of Chengdu Park
   City construction administration (2019MFR0826).

Footnotes

   Disclaimer/Publisher’s Note: The statements, opinions and data
   contained in all publications are solely those of the individual
   author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI
   and/or the editor(s) disclaim responsibility for any injury to people
   or property resulting from any ideas, methods, instructions or products
   referred to in the content.

References