Abstract

Background

   Petal blotch is a unique ornamental trait in angiosperm families, and
   blotch in rose petal is rare and has great esthetic value. However, the
   cause of the formation of petal blotch in rose is still unclear. The
   influence of key enzyme genes and regulatory genes in the pigment
   synthesis pathways needs to be explored and clarified.

Results

   In this study, the rose cultivar ‘Sunset Babylon Eyes’ with rose-red to
   dark red blotch at the base of petal was selected as the experimental
   material. The HPLC-DAD and UPLC-TQ-MS analyses indicated that only
   cyanidin 3,5-O-diglucoside (Cy3G5G) contributed to the blotch
   pigmentation of ‘Sunset Babylon Eyes’, and the amounts of Cy3G5G varied
   at different developmental stages. Only flavonols but no flavone were
   found in blotch and non-blotch parts. As a consequence, kaempferol and
   its derivatives as well as quercetin and its derivatives may act as
   background colors during flower developmental stages. Despite of the
   differences in composition, the total content of carotenoids in blotch
   and non-blotch parts were similar, and carotenoids may just make the
   petals show a brighter color. Transcriptomic data, quantitative
   real-time PCR and promoter sequence analyses indicated that RC7G0058400
   (F3’H), RC6G0470600 (DFR) and RC7G0212200 (ANS) may be the key enzyme
   genes for the early formation and color deepening of blotch at later
   stages. As for two transcription factor, RC7G0019000 (MYB) and
   RC1G0363600 (WRKY) may bind to the promoters of critical enzyme genes,
   or RC1G0363600 (WRKY) may bind to the promoter of RC7G0019000 (MYB) to
   activate the anthocyanin accumulation in blotch parts of ‘Sunset
   Babylon Eyes’.

Conclusions

   Our findings provide a theoretical basis for the understanding of the
   chemical and molecular mechanism for the formation of petal blotch in
   rose.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12870-023-04057-6.

   Keywords: Rose, Blotch, Anthocyanin, Flavonol, Transcriptome

Background

   Petal color patterning, such as flower spots and stripes, is one of the
   most significantly biological and ornamental characteristics for plants
   and has great significance in plant evolutionary biology [[39]1–[40]4].
   As one of the specific floral patterns, petal blotch is found in
   angiosperm families, as observed for example in Cistaceae (e.g., Cistus
   purpureus), Paeoniaceae (e.g., Paeonia rockii), Onagraceae (e.g.,
   Clarkia gracilis), Violaceae (e.g., Viola × wittrockiana Gams.) and
   Compositae (e.g., Senecio cruentus) [[41]5–[42]9]. Although it is
   sometimes associated with elaborated epidermal cell morphologies, color
   patterning is mainly generated by pigment accumulation in the different
   parts of flower petals [[43]3, [44]10, [45]11]. Among four major
   classes of pigments (flavonoids, carotenoids, chlorophylls and
   betaines), flavonoids and carotenoids are widely participated in the
   color formation in most flowers [[46]12].

   Flavonoids include flavones, flavonols, anthocyanins and other
   compounds. Flavonoids are synthesized by a branched pathway that yields
   both colorless compounds (e.g. flavonols) and colored pigments (e.g.
   anthocyanins) [[47]13]. As major pigmented flavonoids, anthocyanins,
   which cause pink, orange, red, scarlet, purple, blue and cyanic flower
   coloration, play a vital role in flower color development. Anthocyanin
   aglycones are divided into six common anthocyanidins, namely cyanidin,
   delphinidin, pelargonidin, peonidin, petunidin, and malvidin
   [[48]14–[49]16]. Flavonols and flavones, as colorless flavonoids, play
   important roles in coloration by co-pigmentation effects with
   anthocyanins in floral organs [[50]17]. Carotenoid is the generic term
   for carotenes and xanthophylls, which provide colors ranging from
   yellow to orange in ornamentals [[51]12, [52]14].

   Pigment (anthocyanin, flavonol and carotenoid) pathways and genes have
   been extensively characterized in model and non-model plants [[53]12,
   [54]18–[55]21]. It was reported that the synthesis of anthocyanin and
   flavonol shares the same upstream pathway as the formation of
   dihydrokaempferol and dihydroquercetin, followed by downstream branch
   for the formation of anthocyanins and flavonols. In this comprehensive
   synthesis process, the key enzymes have been well characterized,
   including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone
   3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid
   3′,5′-hydroxylase (F3′5′H), flavonol synthase (FLS), dihydroflavonol
   4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-flavonoid
   glucosyltransferase (UFGT) [[56]12, [57]22]. The carotenoid
   biosynthesis pathway involves multiple enzymes like phytoene synthase
   (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS),
   carotenoid isomerase (CRTISO), ε-ring cyclase (LCYE) and β-ring cyclase
   (LCYB) contribute to the synthesis of carotenes [[58]12, [59]14].
   Carotenes are catalyzed to produce various xanthophylls by enzymes such
   as ε-ring hydroxylase (CHYE), β-ring hydroxylase (CHYB), zeaxanthin
   epoxidase (ZEP), violaxanthin de-epoxidase (VDE) and neoxanthin
   synthase (NSY) [[60]23].

   Regulation of flavonoid pathways is mostly coordinated by a
   transcription factor complex consisting of R2R3 MYB transcription
   factors (TFs), basic helix–loop–helix (bHLH) TFs and WD40 proteins (MBW
   complex), which activates transcription biosynthetic genes in many
   plants, such as Arabidopsis thaliana, Petunia hybrida and Rosa hybrida
   [[61]13, [62]24–[63]26]. MBW complex also functions in carotenoid
   biosynthesis in Medicago truncatula [[64]27]. Besides MYB, bHLH and
   WD40, many transcription factors are implicated in the control of the
   flavonoid biosynthetic pathway. For example, WRKY transcription factors
   play a role as a flavonoid regulator in Petunia [[65]28]. NAC
   transcription factors are proved to be the necessary partner TFs for
   the anthocyanin biosynthesis in blood-fleshed peach [[66]29]. DOF
   transcription factors have the negative influence on flavonoid
   biosynthesis in Arabidopsis thaliana [[67]30].

   Roses are among the most commonly cultivated ornamental plants
   worldwide and have gained the title of the world’s favorite flower
   [[68]31]. The genus Rosa contains approximately 200 species and over
   40,000 cultivars [[69]32]. R. persica stands out from all wild roses by
   the chestnut red blotch at the base of each yellow petal [[70]33,
   [71]34]. Since Jack L. Harkness took up the persica line of breeding in
   1960s and introduced one of Hulthemia hybrids in 1985 under the name
   ‘Tigris’, the success of hulthemia breeding has been a breakthrough in
   rose breeding in recent years [[72]35]. Hulthemia hybrids with variable
   colors provide a good mode for studying pigment biosynthesis and
   pattern formation (Fig. [73]1). Previous studies report that rose
   petals are known to contain anthocyanins based on cyanidin,
   pelargonidin and peonidin as well as carotenoids. Roses do not produce
   flavones whereas two predominant flavonol aglycones, namely kaempferol
   and quercetin, exist in rose petals [[74]36–[75]41]. Yellow roses have
   been used for research on carotenoids in rose petals [[76]42, [77]43].
   Admittedly, there are various reports on rose petal coloration
   [[78]44–[79]46]. Nevertheless, the mechanism for pigmentation
   patterning of hulthemia hybrids has never been reported.

Fig. 1.

   Fig. 1
   [80]Open in a new tab

   Variable Hulthemia hybrids. a Rosa ‘Sunshine Babylon Eyes ‘. b Rosa
   ‘Xizixiawu’. c Rosa ‘Bull’s Eye ‘. d Rosa ‘Eyes for you ‘

   In this study, the hybrid hulthemia cultivar ‘Sunset Babylon Eyes’ was
   used to explore the pigmentation regulatory network of blotch and
   non-blotch parts during the development of rose flowers. We reported
   the metabolic profiling of flavonoids and carotenoids, as well as gene
   expression dynamics for the blotch and non-blotch petals of five
   different coloring stages using integrated analysis of the metabolome
   and transcriptome. With this extensive analysis of multiple approaches
   in hybrid hulthemia cultivar ‘Sunset Babylon Eyes’, we reveal changes
   in the key pigments and related biosynthesis genes that are associated
   with petal blotch formation.

Results

Flower colors of ‘sunset Babylon eyes’ at different developmental stages

   We collected materials at five stages depending on the progress of
   anthesis and development of blotch [[81]8, [82]47, [83]48]. At S1
   (colorless bud petal stage), petals were yellow-green without blotches.
   Cerise blotches appeared at the base of the light yellow petals at S2
   (initially colored bud petal stage). At S3 (colored bud petal stage),
   blotches grew to about half of the whole petal and turned rose-red
   while non-blotch parts became butter yellow. Blotches grew continuously
   and their color turned crimson while the non-blotch parts became bright
   yellow at S4 (initiating blooming stage). At S5 (blooming stage),
   non-blotch parts were yellow while blotches turned dark red
   (Fig. [84]2a).

Fig. 2.

   [85]Fig. 2
   [86]Open in a new tab

   Petal blotch development and pigments accumulation in rose. a
   Phenotypes of different developmental stages of rose petal blotch. b
   Flower color distribution of Rosa ‘Sunset Babylon Eyes’. c Pigment
   accumulation in rose petal, reflecting the total content of
   anthocyanins (TA), carotenoids (TC), flavone and flavonol (TF) in the
   petal blotch and non-blotch regions during different developmental
   stages. d Flavonol accumulation in rose petal, including kaempferol
   (Total Km) and quercetin (Total Qu). Three independent biological
   experiments were performed. Values represent means ± SE

   To precisely evaluate the color of rose flowers, color parameters L^*,
   a^* and b^* of CIEL^*a^*b^* color system of petals were measured.
   Significant differences were observed in color parameters among the
   blotch and non-blotch parts at different stages (Fig. [87]2b). L^*
   value of blotch and non-blotch parts declined from S1 to S2. From S2 to
   S5, L^* value of the non-blotch parts increased whilst that of the
   blotch parts decreased. Parameter a^* represents green and red color
   from negative value to positive value. The a^* value of non-blotch
   parts saw a slight increase from S1 to S4, and then declined from S4 to
   S5. The a^* value of blotch parts witnessed a fluctuation trend of
   ‘increase-decrease-increase’ from S1 to S5 and stood at the peak at S3.
   Parameter b^* represents blue and yellow color from negative value to
   positive value. The b^* value of the non-blotch parts increased
   continuously from S1 to S4 and then dropped substantially. Conversely,
   the b^* value of the blotch parts experienced an opposite trend.

Identification and quantification of flavonoids in petals of ‘sunset Babylon
eyes’

   Only one anthocyanin: cyanidin 3,5-O-diglucoside (Cy3G5G) was found in
   the blotch parts from S2 to S5 (Table [88]1, Supplementary Fig. [89]1a,
   Supplementary Table S[90]1). Anthocyanins were not detected at S1 and
   in the non-blotch parts from S2 to S5. The content of Cy3G5G was very
   low in the blotch parts at both of S2 and S3. And the total content of
   anthocyanin (TA) in the blotch part at S4 was the highest, which was
   about 7.4 times higher than that of the blotch parts at S2. TA in the
   blotch part at S5 was close to but slightly lower than that at S4 (Fig.
   [91]2c).

Table 1.

   Identification of flavonoids
   Peak no. Retention time (min) λ[vis-max] (nm) λ[vis-acyl] (nm)
   ESI^−MS^−(m/z) Aglycone Main identified molecule References Standard