Abstract

Background

   The Rhododendron sanguineum complex is endemic to alpine mountains of
   northwest Yunnan and southeast Tibet of China. Varieties in this
   complex exhibit distinct flower colors even at the bud stage. However,
   the underlying molecular regulations for the flower color variation
   have not been well characterized. Here, we investigated this via
   measuring flower reflectance profiles and comparative transcriptome
   analyses on three coexisting varieties of the R. sanguineum complex,
   with yellow flush pink, bright crimson, and deep blackish crimson
   flowers respectively. We compared the expression levels of
   differentially-expressed-genes (DEGs) of the anthocyanin / flavonoid
   biosynthesis pathway using RNA-seq and qRT-PCR data. We performed
   clustering analysis based on transcriptome-derived Single Nucleotide
   Polymorphisms (SNPs) data, and finally analyzed the promoter
   architecture of DEGs.

Results

   Reflectance spectra of the three color morphs varied distinctively in
   the range between 400 and 700 nm, with distinct differences in
   saturation, brightness, hue, and saturation/hue ratio, an indirect
   measurement of anthocyanin content. We identified 15,164 orthogroups
   that were shared among the three varieties. The SNP clustering analysis
   indicated that the varieties were not monophyletic. A total of 40
   paralogous genes encoding 12 enzymes contributed to the flower color
   polymorphism. These anthocyanin biosynthesis-related genes were
   associated with synthesis, modification and transportation properties
   (RsCHS, RsCHI, RsF3H, RsF3′H, RsFLS, RsANS, RsAT, RsOMT, RsGST), as
   well as genes involved in catabolism and degradation (RsBGLU, RsPER,
   RsCAD). Variations in sequence and cis-acting elements of these genes
   might correlate with the anthocyanin accumulation, thus may contribute
   to the divergence of flower color in the R. sanguineum complex.

Conclusions

   Our results suggested that the varieties are very closely related and
   flower color variations in the R. sanguineum complex correlate tightly
   with the differential expression levels of genes involved in the
   anabolic and catabolic synthesis network of anthocyanin. Our study
   provides a scenario involving intricate relationships between genetic
   mechanisms for floral coloration accompanied by gene flow among the
   varieties that may represent an early case of pollinator-mediated
   incipient sympatric speciation.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12870-021-02977-9.

   Keywords: Anthocyanin synthesis, Comparative transcriptomics, Flower
   coloration, Gene expression, Rhododendron sanguineum complex, Sympatric
   speciation

Background

   The remarkable diversity of flower colors, especially in wild plants
   has fascinated botanists, ecologists, and horticulturists for centuries
   [[45]1–[46]3]. The coloring of floral organs, a remarkable character of
   flowering plants, is a striking feature of the angiosperm radiation
   [[47]4, [48]5]. Flower color diversity is recognized to be one of key
   adaptive traits correlated predominantly with pollinators (e.g.
   insects, birds) and animals for seed dispersal [[49]6, [50]7].
   Moreover, the flower color phenotype is an important feature for plants
   used for their classification by taxonomists. However, flower color
   appears evolutionarily to be one of the most labile traits, down to
   populations in the same species [[51]7, [52]8].

   The cellular compounds of flowers that contribute to the color profile
   and visually perceived by humans are generally referred to as
   “pigments”. A group of secondary metabolites belonging to flavonoids
   are the main determinants of pigments for coloration in plants, where
   anthocyanins are responsible for red orange to red, purple to violet
   pigments found in flowers, leaves, fruits, seeds and other tissues
   [[53]9, [54]10]. Anthocyanins are the predominant compounds of floral
   coloration, existing in over 90% of angiosperms [[55]11]. The flavonoid
   biosynthetic pathway leading to accumulation of anthocyanins is highly
   conserved and well characterized, and has been extensively studied in
   many species, most of which are in model plants or agriculturally and
   horticulturally important plants [[56]12–[57]15]. Few studies have
   examined the molecular basis underlying the formation and accumulation
   of anthocyanin in wild species [[58]16, [59]17]. Based on these
   studies, three major associated factors have been proposed to be
   involved in anthocyanin accumulation, including transcription
   regulatory genes (MYB-bHLH-WD40 complex) that occur in the nucleus,
   structural genes (CHS, FLS, DFR, ANS) acting in the biosynthetic
   pathway, and transporter genes (GST) transferring anthocyanin from the
   cytosol into the vacuole [[60]10, [61]18, [62]19]. The expression of
   these genes could also be affected by natural variation in sequences
   and cis-regulatory elements as well as epigenetic modifications (such
   as DNA methylation) in the promoter regions [[63]18, [64]20]. Moreover,
   the color of flowers can be stabilized and enhanced by co-pigmentation
   of anthocyanins by flavonols, where it is observed as hyperchromic
   effect, in which the intensity of an anthocyanin content is fortified
   [[65]21]. For instance, the DFR gene along with the FLS gene can
   compete for a substrate leading to the production of different
   anthocyanins and flavanols through two primary branches [[66]22,
   [67]23], thus resulting in co-pigmentation. In contrast to the
   biosynthesis pathways, knowledge of anthocyanin catabolism in plants is
   limited. Some catabolic genes like BGLU and PER have been shown to be
   responsible for anthocyanin degradation [[68]24, [69]25]. Nevertheless,
   the molecular mechanism regulating anthocyanin synthesis has been shown
   to vary among plant species resulting in structural diversity of
   anthocyanins, because the biosynthesis pathway is regulated by multiple
   factors through regulatory networks [[70]26].

   Color is a form of electromagnetic radiation in the range of the
   visible spectrum. The wavelengths reflected by pigments determine the
   color of a flower [[71]27]. Color can be defined and classified in
   terms of Brightness (the intensity of a signal, B), Saturation (the
   purity of a color, S) and Hue (the spectral descriptor of color, H),
   and those features are commonly used to distinguish colors [[72]27,
   [73]28]. Brightness refers to the color intensity that is determined by
   the amount of anthocyanin [[74]29, [75]30], and different color
   component combinations such as B/H, S/H were found to be significantly
   correlated with anthocyanin content as well [[76]31]. Liu et al.
   [[77]32] proposed that the color brightness decreased as the total
   anthocyanin content increased. It was also demonstrated that a
   correlation exists between the saturation/hue ratio (S/H) and
   anthocyanin content [[78]31]. With these parameters, the anthocyanin
   content can be rapidly and non-destructively determined.

   In evergreen azaleas (Rhododendron), anthocyanins and flavanols are the
   main flower pigments, and especially the composition of anthocyanin
   constituents (i.e. cyanidin, delphinidin, malvidin, pelargonidin,
   peonidin, and petunidin), and their quantities determine their flower
   color that ranges from light pink to violet [[79]11, [80]33]. Some
   studies have reported that R. kiusianum with purple-colored flowers
   contain derivatives of both anthocyanidins cyanidin and delphinidin,
   whereas the red-colored flowers of R. kaempferi contain only cyanidin
   derivatives [[81]34]. Le Maitre et al. [[82]35, [83]36] studying Erica
   species, belonging to the same family Ericaceae as Rhododendron, used
   qRT-PCR and UPLC-MS, unraveled the anthocyanin genetic network of
   floral color shifts between red or pink and white or yellow flowered
   species and found losses of single pathway gene expression, abrogation
   of the entire pathway due to loss of the expression of a transcription
   factor or loss of function mutations in pathway genes resulted in
   striking floral color shifts.

   Here, we investigated the genetic basis of flower coloration using a
   highly color polymorphic Rhododendron sanguineum complex. The complex
   (R. subgen. Hymenanthes) includes plants with yellow to pink or crimson
   to blackish crimson flowers that are classified into six varieties
   mainly based on their flower color differences [[84]37]. Members of
   this complex are basically located at high elevations (> 3000 m)
   associated with snow cover [[85]37]. They are endemic to northwest
   Yunnan and southeast Tibet, one of the global biodiversity hotspots
   [[86]38]. This region is also recognized as one of the centers for
   diversification and differentiation of Rhododendron [[87]37, [88]39].
   The flower color polymorphisms of this genus have been traditionally
   viewed as an ecologically adaptive trait that is essential in
   attracting specific pollinators [[89]40–[90]42], and may also be the
   response to environmental variation, such as UV radiations at different
   elevations, temperatures, and soil conditions [[91]32]. Although there
   are studies published on the anthocyanin components and contents in
   Rhododendron flowers, most were solely dedicated to the identification
   of the pigment constituents in the petals of some wild and cultivated
   azaleas using thin-layer chromatography (TLC) and high-performance
   liquid chromatography (HPLC) [[92]11, [93]33]. No study so far focused
   on the molecular mechanisms underlying infraspecific color
   polymorphisms in Rhododendron. The study of closely related entities
   such as a species complex has the advantage of a fairly homogeneous
   genetic background where flower color genes vary and cases of homoplasy
   are limited. Previous studies mainly focused on color shifts at
   different developmental stages of a single species [[94]14, [95]18], or
   covered a number of related species [[96]26, [97]35].

   In the present study, we combined transcriptome sequencing (RNA-seq)
   and genome resequencing with reflectance spectra analyses to elucidate
   molecular and anthocyanin content differences among three differently
   colored naturally occurring varieties of the R. sanguineum complex,
   with yellow flushed pink to deep blackish crimson colored flowers. We
   aimed at studying the correlation between infraspecific flower color
   variation and the expression of candidate genes of the anthocyanin /
   flavonoid biosynthesis pathway. Our findings may allow the proposal of
   a hypothesis for the genetic mechanism of the expression of flower
   color variation and a representative case of pollinator-mediated
   incipient sympatric speciation in the R. sanguineum complex. In
   addition, it is the first study to compare transcriptome profiles in a
   natural system of a non-model species of Rhododendron.

Results

Reflectance spectra and color morph differences

   The sampled individuals can be grouped into three color categories
   representing R. sanguineum var. sanguineum (RsS) with bright crimson
   flowers (Fig. [98]1d), R. var. haemaleum (RsH) where the flowers were
   deep blackish crimson (Fig. [99]1e), and R. var. didymoides (RsD) with
   yellow flushed pink tubes and red petals (Fig. [100]1f). The
   reflectance spectra of the three color morphs varied distinctively
   across the wavelength range, especially between 400 and 620 nm
   (Fig. [101]2a). RsS showed one discrete peak in the red spectrum
   (~ 620 nm), while RsH had a peak further into the far red range
   (> 700 nm). The reflectance of RsD increased across the blue to yellow
   spectrum and peaked in the orange-red range (~ 600 nm) (Fig. [102]2a).
   The flower colors of the three varieties exhibited marked differences
   in brightness, saturation and hue. RsD had the highest brightness
   values followed by RsS, and RsH with the lowest values, while RsH had
   the highest saturation, then RsS with RsD the lowest (Fig. [103]2b, c).
   The hue values were highest in samples of RsD, then RsS and RsH
   (Fig. [104]2b). The ratio of saturation and hue (an indirect
   measurement of anthocyanin content) of RsH was the highest compared to
   those of the RsD and RsS samples (Fig. [105]2d). The ratio values of
   the RsD samples varied much wider than those of the other two varieties
   that maybe linked to variation in the color composition of its
   bicolored flowers (Fig. [106]1).

Fig. 1.

   [107]Fig. 1
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   Morphology and sampling stages of the three different flower morphs
   found in the Rhododendron sanguineum complex in the wild. a Habitat of
   the sampling site; sampling stages of the two tissues for RNA-seq,
   including late flower bud (b) and leaf bud (c) of the varieties (left
   to right) R. sanguineum var. sanguineum, R. var. haemaleum, and R. var.
   didymoides; open flowers of R. var. sanguineum (d), R. var. haemaleum
   (e), and R. var. didymoides (f)

Fig. 2.

   [109]Fig. 2
   [110]Open in a new tab

   Comparisons of the reflectance spectra of three varieties of the
   Rhododendron sanguineum complex. a Reflectance spectra were measured at
   the midpoint of the corolla tubes of the three varieties. b
   Three-dimensional plots of the visible spectra that classified by
   brightness (x-axis), hue (y-axis), and saturation (z-axis) of the three
   varieties. c Boxplots of brightness values of the reflectance spectra
   of the three varieties. d Boxplots of the ratios of saturation over hue
   from reflectance spectra for the three varieties. Boxplots show the
   median, and the box edges represent the 25th and 75th percentiles of
   values for a total of 15 individuals for each variety. Statistical
   significance was determined by a two-tailed Student’s t test. The
   significant differences are noted as asterisks (*P < 0.05; **P < 0.01;
   ***P < 0.001). Red represents R. var. sanguineum (RsS); Purple
   represents R. var. haemaleum (RsH); Yellow represents R. var.
   didymoides (RsD)

De novo transcriptome assembly and quality assessment

   We sequenced a total of 18 RNA-seq libraries from two tissues (flower
   buds and leaf buds) for three individuals for each of the three
   varieties in the R. sanguineum complex using Illumina paired-end
   sequencing. After quality control, approximately 655.8 million (M)
   clean reads (~ 96 gigabase pairs, Gbp) remained with a very uniform
   number of reads between the libraries ranging between 32,111,674 and
   43,353,842 (Table [111]S1). The contig N50 values for the CORSET across
   the three varieties had similar lengths and ranged from 903 to 1125
   base pairs (bp) and the numbers of transcripts ranged from 117,976 to
   171,725 (Table [112]S1). The reads of the 18 individual libraries were
   aligned by mapping the reads back to their variety-specific references,