Introduction

Rose (Rosa damascena Mill.) is a cultivar of rose growing in most parts of Iran and is known for its floral fragrance and excellent color characteristics. It is the most economically valuable rose species in the production of aromatic compounds, which contains high levels of monoterpene alcohols such as geraniol, citronellol, and nerol, as well as phenyl ethyl alcohol1. The essential oil of R. damascena is a complex mixture of various compounds with a wide range of uses and in the food industry, it prevents food spoilage due to its harmless natural oil and antioxidant, antifungal, and antibacterial properties2. In the aromatic materials and perfumery industries, R. damascena essential oil is used to create higher-quality perfumes3,4. Additionally, due to its unique properties, R. damascena is a suitable choice for studying the different compounds of biosynthesis5.

R. damascena is divided into two main flowering groups; summer damascene and autumn damascene. The latter has white flowers and less fragrance, while the former has more popularity, more use, and higher economic value6,7. In this study, multiple genotypes of R. damascena in Iran are specified, but the exact number is not stated in the given context3. Despite limited information on the regulation of floral fragrance biosynthesis, only a few transcription factors, ODORANT1, EOBII, and PhMYB are involved in the regulation of volatile molecule biosynthesis in flowers8. The most abundant transcription factors in rose are the MYB family9. The MYB1 gene is involved in the biosynthesis of rose fragrance10, regulation of the relative expression of PAR (Phenylacetaldehyde Reductase), and the production of the 2-PE (2-Phenylethanol) component are also involved in the suppression or expression of target genes in color and smell11. Certain studies suggested that MYB is an important TF that regulates anthocyanin synthesis in ornamental flowers12,13,14. Sagawa et al.15 stated that a transcription factor called R2R3-MYB in the Mimulus lewisii plant plays an essential role in carotenoid metabolism.

The GGPPS (Geranylgeranyl diphosphate synthase) gene may be involved in the biosynthesis of volatile monoterpenes in the flowers of R. damascena. This gene can be important for adjusting the secondary metabolism of the aromatic components of R. damascena16. The CCD1 (Carotenoid Cleavage Dioxygenase 1) gene pathway is closely related to the biosynthesis of volatile compounds17. Increasing the expression of the CCD1 gene in the petals leads to discoloration and an increase in the amount of ketone compounds in the essential oil11. The CER1 (ECERIFERUM1) gene is an aldehyde decarboxylase encoder18, resistant to cold and drought stress, and the up regulation of the expression of this gene corresponds to an increase in the content of alkanes19. This gene is involved in the synthesis of the precursor aromatic compounds (VLC (very-long-chain) fatty acids) in the endoplasmic reticulum (ER)20.

ANS and RhGT1 are key genes in anthocyanin biosynthesis19. Glycosylation is a prerequisite for anthocyanin biosynthesis, and RhGT1, as a new glycosyltransferase, shows a novel pathway for anthocyanin biosynthesis21. Anthocyanin and other flavonoid components are produced through the phenylpropanoid pathway. Phenylalanine, the main precursor of flavonoids, is converted into required derivatives by the enzyme Phenylalanine ammonia-lyase (PAL). Dihydroflavonols can be transformed into flavonols and anthocyanins. The production of flavonols is catalyzed by the enzyme flavonol synthase (FLS), while dihydro flavonol 4-red case (DFR) is the first enzyme that takes part in the step-by-step cascade of anthocyanin synthesis. Stabilization and alteration of anthocyanin molecules affect the final color of the flower22. In addition, ANS gene is a major gene in anthocyanin biosynthesis, and overexpression of this gene may increase anthocyanin pigments in petals11. The main secondary metabolites of R. damascena are phenolic acids (gallic, caffeic, chlorogenic, and coumaric) and flavonoids (rutin, quercetin, kaempferol, and apigenin)23. Kanani et al. reported that the highest content of flavonoids was estimated in the fully open flower stage. In addition, the activity of PAL enzyme showed a positive association with the production of some flavonoids24.

Jasmonic acid (JA) and its derivatives are key messenger molecules and play an important role in many biological processes, such as growth inhibition, senescence, and plant defense25. MeJA has been used to increase the production of secondary metabolites by inducing defense responses in many species, such as volatile terpenoids in Amomum villosum26, triterpenes in Euphorbia pekinensis27 and Tropane alkaloids in Hyoscyamus niger28.

With these considerations in mind, the current study aimed to investigate the effectiveness of the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathway genes in the biosynthesis of aromatic compounds and the coloration of roses during two critical stages of flower development: bud and fully open flower. The study examined the effects of two different concentrations of MeJA on these processes. Additionally, the relative expression levels of the PAL, PAR, FLS, MYB1, CER1, GT1, CCD1, ANS, and GGPPS genes were measured in the treated samples. To gain a deeper understanding of the changes occurring during flower growth, gas chromatography-mass spectrometry (GC–MS) and real-time PCR techniques were employed to analyze the phytochemical and molecular alterations.

Materials and methods

Plant materials

The petal samples were collected from two morphs of R. damascena, in two colors, white and Hot pink, grown at the research station on the Agricultural Campus of the University of Tehran (35°48'N, 51°23'E, elevation 1305 m). Flower buds were harvested at two important flower developmental stages (S1 = budding stage, S2 = full opening stage) )Table 1). Both white and Hot pink morphs of R. damascena were grown under identical environmental conditions (mean temperature 25 ± 2°C, relative humidity 60 ± 5%, and 16/8 h light/dark photoperiod) in the orchard section of the faculty's farm. Uniform cultivation practices, including drip irrigation (4 L/plant/day), fertilization (NPK 20:20:20, 5 g/plant/month), and integrated pest management, were applied to both rose types. These measures ensured consistent climatic conditions and minimized abiotic stress differences between the morphs throughout the study period.

Table 1 Characteristics R. damascena in two stages of flower growth (S1 = bud, S2 = full open flower).
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To investigate the effect on gene expression, MeJA treatment was applied at two concentrations: 0 μM (control) and 300 μM, both prepared in a 0.1% (v/v) ethanol solution. Control plants were sprayed with distilled water containing 0.1% (v/v) ethanol. The solutions were applied using a handheld sprayer (2 mL per plant) at dawn to minimize evaporation. Samples were collected 48 h post-treatment. The harvested petals were immediately placed in sterile paper bags and transported to the laboratory in an aseptically maintained portable cooler at 4°C for essential oil extraction and physiological evaluation. For molecular analyses, petal samples were flash-frozen in liquid nitrogen on-site and subsequently stored in an ultra-low temperature freezer (Thermo Scientific™ Forma™ 88,000 Series, -80°C) for future processing 29,30,31.

Determination of total flavonoid content (TFC)

Total Flavonoid Content (TFC) was calculated based on the method introduced by Khorasani Esmaeili et al. (2015) with slight modifications. Solutions of 4% NaOH, 5% NaNO2, and 10% AlCl3 in distilled water were used to test. AlCl3 solution was prepared by gradually adding AlCl3 to distilled water under a the the Laminar Hood (Model NU-437-400E, NuAire, USA) carefully and steadily32. 25 μL of each sample solution, 100 μL of distilled water, and 7.5 μL of 5% NaNO2 solution (in eight replicates) were added to a 96-well plate (Corning, Catalog Number 3596). After 6 min, 7.5 μL of 10% AlCl3, 100 μL of 4% NaOH, and 0 μL of distilled water were added to each well. The plate was covered with aluminum foil, and the absorbance of the solutions was read after 15 min using a spectrophotometer. Absorbance was measured at 510 nm with an ELISA microplate reader (Model 680, Bio-Rad, Software version 1.2). TFC was expressed as mg of rutin equivalents (CTE) per gram of extract dry matter, using a calibration curve prepared with concentrations ranging from 0 to 100 μg/mL.

Determination of total anthocyanin content (TAC)

100 mg of petal tissue was dissolved in 100 ml of acidic methanol (methanol and hydrochloric acid at a volume ratio of 99:1) using a porcelain mortar and pestle. The desired solution was kept in the dark for 24 h at a temperature of 25°C. Subsequently, it was centrifuged (Hettich Universal 320R) for 15 min at 4000 rpm, and the absorbance of the supernatant was measured at a wavelength of 530 nm using a Thermo Scientific™ Evolution 201 UV–Visible spectrophotometer with Vision Pro Software version 4.0. Using the formula, A = εbc and considering the extinction coefficient (ε) equal to 33,000 \({\text{mol}}^{2}{\text{cm}}^{-1}\), concentration of the results was presented in μM per gram of fresh weight. In this formula, A = absorption of the high solution, b = cuvette path length, which is equal to 1, and c = concentration of anthocyanin per mole of fresh weight of the sample33.

Essential oil (EO) extraction and gas chromatography-mass spectrometry (GC/MS) analysis of the EOs compounds

To extract the essential oil by hydro-distillation method, 250 g of fresh petals were separated using Clevenger Apparatus (Quickfit, England) according to the method recommended by34. Then, the volatile oil layer on top of the aqueous layer was collected in a glass vial. The amount of essential oil was obtained based on the percentage ratio (v/w). The percentage of each component of the essential oil was determined using The GC–MS instrument, Agilent 7890A gas chromatograph coupled with a 5975C mass spectrometer detector (Agilent Technologies, USA), equipped with a mass spectrometer detector and CTC CombiPAL for liquid and headspaces. In the present study, a silica column (Resteck HP-5MS) made of 5% phenyl methyl silox and injector temperature of 325 °C were used with the following features: 60 m length, 320 µm diameter, and 0.25 µm of particle size. The injector ran in the splitless mode with a temperature of 280 °C. The temperature of the oven was set at 50 °C for 2 min, following an increased slope of 3 °C min−1 to 240 °C for 12 min, and then held to 290 °C at a rate of 20 °C min−1. The chromatographic separation was performed using a specific solvent system with a delayed solvent peak, resulting in a total run time of 79.833 min. The carrier gas was helium (purity 99.999%) flowing at a rate of 1.0 mL min−1. Peaks were analyzed with Mass lab software at one scan per second speed. Using the calculation of the Quartz coefficient (injection of normal n-alkane hydrocarbons (c11-c28) under the same conditions as the injection of essential oils), identifying essential oil compounds and checking the mass spectrum proposed by the GC–MS libraries, including "NIST05a.L & wiley7n.l ", were compared 35,36,37.

Extraction of ribonucleic acid (RNA), cDNA synthesis, and design of primers

Extraction of total ribonucleic acid from the tissues of young flower petals and buds was conducted using the manual CTAB method according to the slightly modified instructions38 and was treated with DNaseI (Qiagen, Germany) to remove genomic contamination. The quantity of RNA was assessed using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, USA), and the quality was evaluated by 1% agarose gel electrophoresis. To synthesize cDNA from RNA, Parstos cDNA synthesis kit (Pars Tous Biotechnology, Iran) was used Primers were designed for the Actin gene (as reference gene: KT965025.1), using Primer3 software (version 4.1.0). The proposed primers were evaluated using Oligo Analyzer online (Integrated DNA Technologies, USA) software to have key features (Self-Dimer, Hetero-Dimer, and Hairpin) and primer binding temperature. The pair of primers for the actin gene as a housekeeping gene and the primer for the studied genes are given in Table 2.

Table 2 Characteristics of primers used in molecular expression by qRT-PCR.
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Real-time PCR analysis

The SYBR® Green Real-Time PCR Master Mix kit (Parstous, Iran), which has Syber-green, fluorescent dye, was used to perform the qRT-PCR test. The real-time PCR reaction was performed using the Rotor Q-gene device (Qiagen, Germany), according to the instructions kit. The Actin housekeeping gene was considered as internal control and for data normalization. The reaction program included 95°C for 15 min and 40 cycles: 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s. All reactions were mixed, each with two technical replicates for statistical analysis, and the relative expression of genes was calculated using the formula39.

Plotting heatmap and dendrogram

In this study, cluster analysis was conducted to investigate the relationship between physiological parameters and genetic variables. For clustering, the heatmap package (version 3.1.3) in the R software (version 4.4.1) was used, and the distance matrix was calculated using the Ward method40. Information related to genes was placed in columns and samples in rows. Row clustering was represented with a dendrogram, and subsequently, different colors were assigned to genes to display expression values. Ultimately, a heatmap was drawn based on the data matrix and clusters. Colors were adjusted based on data values to make patterns and differences easily observable41.

Result

Extraction of essential oils and evaluation of volatile organic compounds (VOCs)

The essential oil extracted from 250 g of fresh R. damascena flowers had a delightful smell, which plays a significant role in attracting insects for pollination and is used in the perfumery and cosmetics industries. Separation and identification of essential oil compounds were conducted using GC/MS analysis. The Total Ion Chromatogram (TIC) of both white and Hot pink R. damascena flowers (Fig. 1) offers a comprehensive profile of the volatile compounds detected in the samples. In general, 26 compounds were identified in four samples (Table 3). Ten volatile compounds were found in the full opening stage of R. damascena, with the principal components being linalool, nerol, β-citronellol, geraniol, nonadecene, and heneicosane. Geraniol, linalool, and nerol are classified as acyclic monoterpenoids, which are a subgroup of terpenoids characterized by their linear structure without cyclic rings. Minor components included α-pinene, geranyl acetate, tricosane, and pentacosane. Although 13 species of monoterpenes and sesquiterpenes were detected, their overall abundance was lower compared to the more prevalent aliphatic components. Only a small number of monoterpenes and sesquiterpenes were found, whereas a large number of aliphatic components were found. The effect of MeJA treatment led to an increase in the volatile compounds such as α-Pinene, geraniol, β-citronellol, nerol, linalool, β-myrcene, β-pinene, sabinene, and geranyl acetate in white flowers, and geraniol and geranyl acetate in pink flowers (Fig. 2). The essential oil components have been classified into two main categories: major and minor, based on two primary criteria: first, the relative abundance of the components in the samples, and second, their impact on the floral aroma profile and industrial applications. Compounds such as linalool, although present in smaller absolute quantities, are categorized as major components due to their significant role in the overall scent of the flower. This classification considers not only the absolute amounts but also the importance of the compounds in various applications such as perfumery and attracting insects for pollination. Since minor components also play a crucial role in completing the aromatic profile and exerting biological effects, a comprehensive analysis of all identified compounds can provide a better understanding of the complex mechanisms underlying floral scent formation.

Fig. 1
figure 1

Total Ion Chromatogram (TIC) of white and Hot pink of R. damascena Mill fresh petals.

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Table 3 Percentage composition of the essential oils isolated from samples of R. damascena petals.
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Fig. 2
figure 2

Total Ion Chromatogram (TIC) of white and HotPink of R. damascena Mill. Fresh petals were treated with methyl jasmonate (MeJA) ) 300 µM for 48 h).

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The results showed that the production rate of monoterpene compounds was 28.32%, sesquiterpene 2.14%, and aliphatic 48.95% in white petals after 48 h of the application of the MeJA. Similarly, the application of this hormone in Hot pink petals led to a decrease of 42.73% in terpenoid compounds and an increase of 40.99% in aliphatic compounds. The highest number of volatile compounds, including linalool (1.68%), β-citronellol (7.98%), and nerol (3.38%), were obtained in pink flowers. Meanwhile, the highest amount of geraniol (23.54%) was obtained from pink flowers under the influence of the MeJA hormone, which may have potential applications in various industries.

Total flavonoid content (TFC)

The total flavonoid content of the essential oil was determined according to42 using the aluminum chloride colorimetric method with slight modifications and rutin as the standard. The results were expressed as mg of rutin equivalents per every gram of dry weight of the plant (mg RE/g Dw). According to the results, the total flavonoid content (Fig. 3) in the Hot pink and white R. damascena petals were 133.20 and 79.30 mg RE/g Dw, respectively, showing a significant difference between the two morph. This difference is clearly demonstrated in Fig. 3, which shows higher flavonoid content in Hot pink petals compared to white petals.

Fig. 3
figure 3

Content of total flavonoid and anthocyanin in white and Hot pink petals of R. damascena.

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Total anthocyanin content (TAC)

The results of the total anthocyanin content showed that the amount of TAC in Hot pink petals (37.48 µmol/g DW) and white petals (2.12 µmol/g DW) were significantly different. While the flavonoid content in pink petals was higher than that of white ones (Fig. 3), it's important to note that flavonoid content and anthocyanin content, although related, are not directly proportional. The amount of anthocyanin in pink petals was 18 times higher than that of white petals, which suggests a complex relationship between total flavonoid content and specific anthocyanin levels. This complexity suggests that although higher levels of flavonoids may be associated with a higher content of anthocyanins, they do not necessarily predict it.

Gene expression analysis

To evaluate the differences among the groups (S1, S2, TS1, and TS2) for each petal color, a one-way ANOVA followed by Duncan's multiple range test was performed. The results of the variance analysis are summarized in Table 4. The results of the ANOVA revealed significant differences among the treatment groups for several dependent variables. Specifically, the mean square values for ANS (11.844), PAR (1.741), CCD1 (310.66), PAL (2.851), FLS (39.623), CER1 (149.84), MYB1 (1258.29), and GT1 (9.453) were significantly higher compared to the error mean squares (1.98, 0.44, 0.1, 2.05, 0.66, 0.64, 9.86, 121.67, 0.91 respectively), indicating that the treatment groups had a significant effect on these variables (p < 0.01 or p < 0.05).

Table 4 Analysis of variance (ANOVA) results for gene expression levels.
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Expression of genes (MYB1, CCD1, GGPPs) involved in fragrance and color in petals in two growth stages

The expression pattern of the CCD1 gene in two different morphs was found to be almost identical. The expression of this gene in ACC.1 was higher than in ACC.26, and the highest amount of expression was recorded for S1 stage under the influence of MeJA stimulus (30-fold increase). A highly significant effect of treatments on CCD1 was observed, with data analysis revealing a substantial interaction effect (P > 0.01) between genotype and different growth stages (S1 and S2). These results were in accordance with the biochemical data, as the number of flavonoids and anthocyanins in ACC.1 petal organ was higher compared to ACC.26. Furthermore, the expression of the CCD1 gene was found to be positively correlated with the number of ketone compounds in the petals. Volatile terpenoids synthesized from carotenoid cleavage contribute significantly to the aroma of roses, particularly in species like Rosa damascena, known for its distinct fragrance used in perfume production. Additionally, carotenoid-derived compounds can influence petal color through their effects43. The analysis of MYB1 gene expression revealed a decrease in ACC.26 and an increase in ACC.1 at the S2 stage compared to the control, suggesting enhanced fragrance and color. The treatments significantly impacted MYB1 levels, indicating a substantial influence of these treatments on this transcription factor. In ACC.1, the TS1 stage exhibited a threefold increase in expression compared to the control (S1), while the TS2 stage showed an eightfold increase. However, the effect of MeJA treatment was not as straightforward as initially interpreted. A comparison between the S2 stage (59-fold increase) and the MeJA-treated TS2 stage (eightfold increase) revealed a decrease in MYB1 expression. This decrease may be attributed to complex interactions between the developmental stage and internal gene regulation mechanisms rather than a direct negative effect of MeJA. It is important to note that the apparent decrease from S2 to TS2 could reflect the natural progression of gene expression during petal development, with MeJA potentially modulating this process rather than simply enhancing expression. There was no significant effect of the treatments on GGPPS, suggesting that the treatments did not significantly influence the GGPPS levels. On the other hand, in ACC.26, the relative expression of the GGPPS gene only experienced a slight rise in the S2 under the influence of MeJA treatment, whereas the effect of MeJA on gene expression was reduced in the first growth stage (Fig. 4).

Fig. 4
figure 4

Quantitative real-time PCR analysis of relative expression level of CCD1, GGPPS, MYB1 (scent and color biosynthetic gene) during flower development (S1 and S2) of two accession numbers (ACC.26 and ACC.1) in R. damascena. Relative expression was calculated based on the threshold cycle (CT) method after normalization to the beta-actin gene. Bud stage (S1) was selected as a reference for white (ACC.26) and Hot pink (ACC.1) petals. Vertical bars represent standard deviations (n = 3). [Abbreviations = S1:bud, S2: Full open flower, TS1: MeJA-treated bud, TS2: MeJA-treated Full open flower]. Error bars represent the values of the mean ± standard error (SE), with n = 3. Different letters (a and b) indicate significant differences (p < 0.05) among S1, S2, TS1, and TS2 for each petal color, as determined by one-way ANOVA followed by Duncan's multiple range test.

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Expression of PAL, CER1, and PAR genes (involved in aroma) during two flower developmental stages

The results of analyzing the gene expression regarding scent in two control conditions (without MeJA treatment) and the conditions treated with MeJA (300 μM concentration 48 h before harvest) in R. damascena showed that in ACC.26 )white flowers(, The highest expression of the PAL gene was observed in the bud stage, with MeJA treatment having no significant effect on gene expression at this stage. In contrast, in ACC.1 (Hot pink petals), PAL gene expression was notably higher in the S2 stage. Moreover, the application of MeJA treatment in both biological stages increased PAL gene expression. Sample TS1 had the highest relative expression (2-folds) compared to the control in the PAR gene. MeJA elicitor was effective in the bud growth stage of ACC.1 while relative expression experienced a decrease compared to the control in other samples. The relative expression level of all CER1 gene samples decreased in comparison with the control, except for sample TS2, which had an increase in the biological stage of fully open flower in Hot pink morph under the influence of MeJA treatment. The frequency of the CER1 gene transcript, which is predicted to function in the synthesis of alkanes, was highly induced (20-Folds) by MeJA treatment (Fig. 5).

Fig. 5
figure 5

Quantitative real-time PCR analysis of relative expression level of PAL, CER1, PAR (aroma biosynthetic gene) during flower development (S1 and S2) of two accession numbers (Acc.26 and ACC.1) in R. damascena. Relative expression was calculated based on the threshold cycle (CT) method after normalization to the beta-actin gene. Bud stage (S1) was selected as a reference for white (Acc.26) and Hot pink (ACC.1) petals. Vertical bars represent standard deviations (n = 3). [Abbreviations = S1:bud, S2: Full open flower, TS1: MeJA-treated bud, TS2: MeJA-treated Full open flower]. Error bars represent the values of the mean ± standard error (SE), with n = 3. Different letters (a and b) indicate significant differences (p < 0.05) among S1, S2, TS1, and TS2 for each petal color, as determined by one-way ANOVA followed by Duncan's multiple range test.

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Expression of FLS, GT1, and ANS genes (involved in petal color) in two growth stages

Anthocyanin, synthesized by the ANS gene, plays a crucial role in the coloration of Rosa damascena. In the S2 developmental stage without elicitor application, the relative expression of ANS significantly increased in the ACC.1 sample, reaching a peak with a sixfold elevation. Upon elicitor application during the TS2 stage, the relative expression of ANS increased twofold compared to the control in ACC.1. In contrast, the ACC.26 sample showed consistently low relative expression of the ANS gene across all developmental stages, with minimal variation. These molecular findings align with biochemical data, indicating that ACC.1, characterized by Hot pink petals, exhibits higher anthocyanin content relative to ACC.26. Additionally, the peak relative expression of the FLS gene was observed in the TS2 sample, showing an 11-fold increase. Analysis of the data demonstrated elevated FLS expression levels during the fully open flower growth stage in response to MeJA in ACC.1 (Hot pink morph). However, in ACC.26 (white morph), the expression of FLS was not enhanced by MeJA treatment. There was a highly significant effect of the treatments on FLS, indicating a substantial impact of the treatments on this variable in ACC.26. A comparison between samples Acc.26 and ACC.1 revealed differential expression levels of the GT1 gene during the S2 growth stage, with higher expression observed in the Hot pink morph (ACC.1) and lower expression in the white morph (Acc.26). The treatments had a significant effect on GT1 gene expression during stage S2, indicating a substantial influence on the gene's expression during this developmental stage. Specifically, MeJA treatment induced a positive and incremental effect exclusively in ACC.1 during stage S2, where gene expression progressively increased across stages S2, TS1, and TS2, particularly enhanced by MeJA. In contrast, gene expression in Acc.26 during stage S2 showed a slight decrease and minimal change under MeJA treatment. It is important to note that the expression of GT1 was not affected by MeJA treatment in the white morph (ACC.26) at any developmental stage (Fig. 6).

Fig. 6
figure 6

Quantitative real-time PCR analysis of Relative expression level of FLS, GT1, ANS (color biosynthetic gene) during flower development (S1 and S2) of two Accession numbers (Acc.26 and ACC.1) in R. damascena. Relative expression was calculated based on the threshold cycle (CT) method after normalization to the beta-actin gene. Bud stage (S1) was selected as a reference for white (Acc.26) and Hot pink (ACC.1) petals. Vertical bars represent standard deviations (n = 3). [Abbreviations = S1:bud, S2: Full open flower, TS1: MeJA-treated bud, TS2: MeJA-treated Full open flower]. Error bars represent the values of the mean ± standard error (SE), with n = 3. Different letters (a and b) indicate significant differences (p < 0.05) among S1, S2, TS1, and TS2 for each petal color, as determined by one-way ANOVA followed by Duncan's multiple range test.

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Correlation and relationship between genes and parameters

Cluster analysis

The heatmap analysis revealed a categorization of the studied genes into two main clusters, prominently visible at the top and bottom. Despite the lack of a clear segregating pattern for these clusters or conditions, the need for enhanced visual analysis was underscored. The first cluster encompassed four genes (GT1, GGPPS, MYB1, and ANS), while the second cluster included five genes (CER1, PAL, CCD1, PAR, and FLS), all grouped based on their high gene expression. The horizontal cluster analysis, comprising five groups, indicated that white (W) and pink (P) petals each corresponded to a distinct cluster, albeit with exceptions. For instance, PS1, representing the bud stage of pink petals, was grouped with white petals, exhibiting low and negligible gene expression, akin to other white petals. An exception was observed in a white petal sample that demonstrated an increase in FLS gene expression, potentially attributable to the influence of the MeJA stimulant. Clustering based on the impact of the MeJA stimulant revealed that the genes FLS, CER1, PAL, CCD1, and PAR all fell within a single group, exhibiting the highest gene expression. In conclusion, the findings underscore the complexity of gene expression regulation in response to MeJA. They also highlight the variability in gene expression patterns among different samples, exemplified by the white petal sample exhibiting an anomalous increase in FLS gene expression. The PS2 sample emerged as the superior specimen, characterized by a substantial upregulation in the expression of genes GT1, GGPPS, MYB1, and ANS. (Fig. 7).

Fig. 7
figure 7

Shows the heatmap and cluster analysis of gene expression in distinct samples of white and pink petals under the influence of applied and unapplied MeJA-treatment. (PS2: Young stage pink flower; PT2: Young stage pink flower by MeJA-treatment ; PT1: Bud stage pink flower by MeJA-treatment ; WTS2: Young stage white flower by MeJA-treatment; WTS1: Bud stage white flower by MeJA-treatment; WS2: Young stagewhite flower; WS1: Bud stage white flowe; PS1:Bud stage pink flower ).

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Discussion

The essential oil compounds of R. damascena were analyzed using GC/MS. Key components included linalool, nerol, β-citronellol, geraniol, nonadecene, and heneicosane, while α-pinene, geranyl acetate, tricosane, and pentacosane were minor. MeJA treatment notably increased α-pinene, geraniol, β-citronellol, nerol, linalool, β-myrcene, β-pinene, sabinene, and geranyl acetate in white flowers, and geraniol and geranyl acetate in Hot pink flowers. Geraniol and linalool are acyclic monoterpenoids, while nerol is an acyclic monoterpenoid alcohol found in numerous plants' essential oils. These compounds are widely used in the cosmetics and perfume industries due to their desirable scent. Valid medicinal properties, such as tumor cell growth inhibition in geraniol, antifungal effect in nerol, and anticonvulsant, antimicrobial, anti-inflammatory, and neuroprotective properties in linalool, have been reported for these compounds44.

Studies have shown that the amount of geraniol varies from 10 to 45 percent, and the climatic conditions, stresses, and growth stages of the plant effectively synthesize this substance in the R. damascena petal organ45,46. In the current study, we perceived a 1.5% increase for geraniol under the effect of elicitor compared to the condition without elicitor in the pink flower. Therefore, it is concluded that this treatment had a positive and increasing effect on the amount of geraniol.

Citronellol is a precursor to the aromatic composition of rose oxide and contributes to the sweet smell of essential oil. This compound is more important than geraniol in the perfumery industry47. A study on the volatile compounds of roses showed that the principal compounds of these oils are citronellol, geraniol, nerol, nonadecane, henicosan, and linalool. The study also demonstrated that the growth and ___location of flowers, i.e., climatic conditions, have a significant impact on the production of essential oils (volatile compounds)48.

The analysis of gene expression in the white morph shows that MeJA markedly increases the expression of FLS and ANS genes, independently of other environmental factors or external stimuli. These genes are pivotal in anthocyanin and flavonol biosynthesis, directly impacting the production and release of VOCs like α-pinene, geraniol, β-citronellol, nerol, linalool, β-myrcene, β-pinene, sabinene, and geranyl acetate49,50,51. This underscores the complex network of plant metabolism essential for coloration, defense, and environmental interactions52. Additionally, MeJA upregulates the expression of CCD1 and MYB1 genes in the pink morph. Specifically, CCD1 and MYB1 are upregulated at both the TS1 (bud stage) and TS2 (fully open flower stage) under MeJA treatment. CER1 is upregulated at the TS2 stage, and PAR is upregulated at the TS1 bud stage. This stimulation also results in elevated concentrations of geraniol and geranyl acetate in pink flowers, thereby supporting the biochemical analyses. These genes and enzymes play essential roles in biosynthetic pathways and regulatory networks that generate essential oil components and other metabolites. They enable diverse plant functions and adaptations to environmental signals and stressors53,54.

The analysis of Trachyspermum ammi L. essential oil compounds showed that the production rate of alpha terpene, the precursor of monoterpenes, increased after 24 h of applying the MeJA hormone. The MeJA elicitor stimulates the biosynthesis pathway of secondary compounds and affects the expression level of the MEP pathway genes one day after the hormone application, which increases the production of monoterpenes55,56,57. Previous research has shown that citronellol, geraniol, nerol, linalyl acetate, and dihydro citronellol acetate are the main components of rose essential oil, and their amount increases during the flower development process. The quantity of these compounds decreases at the end of flowering during flower aging16, which is consistent with the studies conducted during this review.

Anthocyanins, which are one of the six broad groups of plant phenols, are the main flower pigments in higher plants58,59. As flavonoids, these water-soluble pigments have a wide range of biological functions and positive effects on human health60,61. In natural conditions, anthocyanins are often found in the form of anthocyanin glycosides60 and are biosynthesized through the phenylpropanoid pathway, a branch of the shikimate pathway62. The color stability of anthocyanins is influenced by various factors, including the structure of the pigment, pH, temperature, oxygen, light, and water activity. The alteration of the anthocyanin structure is pH-dependent63, and the color change of anthocyanin-containing extracts ranges from purple to red in acidic solutions and from green to yellow in alkaline solutions64. In our experiment, the extract from pink petals was bright red (acidic), while the extract from white petals was yellowish, indicating the presence of a significant amount of anthocyanin in the tested samples. This observation indicates a significant presence of anthocyanins in the tested samples. Additionally, variations in carotenoid content may also contribute to the observed color differences, as carotenoids typically impart yellow to red hues, and their interaction with anthocyanins can further alter the perceived color. Therefore, the color differences observed in the extracts can be attributed to variations in anthocyanin content, the pH of the extracts, and potentially the carotenoid content. According to a report by Zvi et al. (2008), the production of volatile phenylpropanoid/benzenoid compounds in petunia increased tenfold with anthocyanin pigment65. Additionally, the results obtained from Chroho et al. on improving color retention without affecting the gustatory quality of strawberries show that the addition of polyphenols from R. damascena petals leads to an increase in the half-life and a decrease in the thermal degradation of strawberry anthocyanins66.

The Rosa damascena carotenoid cleavage dioxygenase 1 (RdCCD1) protein contributes to the formation of constituents of the rose aroma67. The CCD1 gene C13 nor isoprenoids such as β-damascenone, β-damaskone, and β-ionone are the key flavor compounds in rose essential oil, which are obtained from carotenoid degradation. Additionally, the CCD1 gene generates carotenoid-derived volatiles with fruity fragrance, using carotenoids as substrates, using carotenoids as substrates, through the Carotenoid Cleavage Dioxygenase (CCD) enzymes68. The carotenoid cleavage dioxygenase pathway, which is located downstream of the terpenoid biosynthesis pathway, is closely related to the biosynthesis of volatile compounds17. Transcription factors (TFs) act as the main regulators of cellular processes 69 and control gene expression in plants70. MYB transcription factors play a crucial role in the positive regulation of many secondary metabolites at the transcriptional level, including flower color formation, flavonoid synthesis71, and anthocyanin biosynthesis regulation72. GGPPS is responsible for the production of geranylgeranyl pyrophosphate (GGPP), which provides a platform for post-translational modification (geranylgeranylation) of proteins73. GGPPS is an essential enzyme in the isoprenoid biosynthesis pathway (IBP) and is involved in isoprenoid biosynthesis and metabolism74. GGPPS plays a key role in cytoskeleton regulation, signaling pathways, intracellular transport, and protein prenylation75.

Previous studies have shown that the MYB transcription factor, a key regulator, has the highest expression during petunia's release of flower scent compounds in the full flowering stage76. Additionally, the MYB transcription factor plays an important role in the aroma formation of pear fruit77. Several candidate transcription factors, such as MYB1, have been linked to metabolite flux in the phenylpropanoid pathway, resulting in the production of flavonols/anthocyanin in monocytogenes, which then produces fragrant white flowers of Narcissus and fragrant orange corollas of Narcissus tazetta8. Increasing the expression of the CCD1 gene also increases the amount of ketone compounds in the essential oil78. Earlier studies have shown that with the increase in the expression of CCD1 genes, the petals become colorless, which is consistent with our studies. Therefore, in the white morph , the petals become increasingly pale due to the increase in the growth stage. However, the Hot pink morph the S2 stage, under the influence of MeJA, led to increased gene activity and the relative paleness of the petals. Yu et al. demonstrated that the increase in geraniol content is directly influenced by the high expression of GGPPS in the petals of 'Old Blush' (OB, with intense aroma). The combination of IPP and DMAPP is the primary source for the synthesis of geranylgeranyl pyrophosphate (GGPP), farnesyl diphosphate (FPP), and geranyl pyrophosphate (GPP), which are from the terpene family49. GGPPS functions as a protein that plays a key role in cytoskeleton regulation, signaling pathways, intracellular transport, and protein prenylation 75.

As a key regulator, PAL catalyzes the first step in the biosynthesis of the phenylpropanoid pathway, which is the trans-cinnamic acid production from the deamination of phenylalanine79,80. PAL plays an important role in the biosynthesis of flavonoids81, the synthesis of phenylpropanoid/benzenoid volatiles in flowers82, and the biosynthesis of secondary metabolic pathways in higher plants83. PAR catalyzes the reduction of 2-phenylethylamine to produce 2-phenyl ethanol, a constituent of floral scent in petals84. The advantage of PAR is the reduction of the required ketone substrates, which is done by the regeneration of NADH and the oxidation of secondary alcohols, such as 2-propanol85. CER1, the main gene of the alkane biosynthesis pathway, is an alkane-forming enzyme86,87 that converts VLC aldehydes into alkanes88 and encodes aldehyde decarboxylase89, wax biosynthesis86 and plant flexibility in response to biotic and abiotic stresses90. The CER1 gene encodes a protein localized to the endoplasmic reticulum (ER) and plays a crucial role in the biosynthesis of VLC alkanes, which are integral components of plant cuticular waxes20,91. While the gene product does not directly synthesize aromatic compounds, the resulting long-chain alkanes can contribute to the VOCs profile of flowers. These compounds, although not aromatic per se, can influence floral scent composition and emission patterns49,92. The involvement of CER1 in alkane biosynthesis thus indirectly affects the overall olfactory characteristics of flowers, highlighting the complex interplay between seemingly unrelated metabolic pathways in determining floral volatile emissions. Cuticular wax also consists of VLC aliphatic fatty acid derivatives and aromatic compounds 93. CER1-mediated wax production on the petal organ is necessary for the synthesis of aliphatic components of the pollen wall87 and the "petal effect"94. Previous studies have shown that the CER1 gene leads to the production of 2-phenyl ethanol, which creates a pleasant scent in the petals85,95. The discrepancy between the increased expression of the CER1 gene and the slight increase in alkane content in Hot pink R. damascena following MeJA treatment can be attributed to several factors. First, the regulation of alkane biosynthesis is complex, involving multiple genes and enzymatic steps beyond CER1 expression, including post-transcriptional, translational, and post-translational modifications. Second, the availability of precursor molecules and metabolic flux through the alkane biosynthetic pathway can limit alkane production despite elevated CER1 levels. Third, MeJA's effects on gene expression and metabolite levels are influenced by environmental, physiological, and genetic conditions, which may vary across different experimental setups. Additionally, the presence of inhibitory or competitive pathways might also play a role in modulating the final alkane content. Therefore, the interplay of these factors likely explains the observed non-compliance between CER1 expression and alkane levels17,96,97. The studies demonstrated that the highest PAR transcript peaked in the petal and at the unfurling stage (fully open flower)98; also, PAR is classified as a short-chain dehydrogenase reductase (SDR). Some researchers have revealed that olive (Olea Europaea) is a rich source of bioactive polyphenols that can produce hydroxytyrosol, an important precursor of acetonide99,100,101. In this regard, PAR is one of the genes related to the synthesis of hydroxytyrosol. The results of the above-mentioned phenethylamine transcript study supply a basis for analyzing the acteoside synthesis pathway and extracting regulatory genes and key enzymes.

The ANS gene is a key gene responsible for synthesizing the final stage of anthocyanins. The enzyme encoded by this gene catalyzes the conversion of anthocyanidin precursors into colorless anthocyanidins54,102. Anthocyanin biosynthesis is carried out by a branch of the flavonoid biosynthesis pathway that is also responsible for isoflavonoid and flavonol biosynthesis103 .Glycosyltransferases (GT1) is another key gene involved in anthocyanin biosynthesis19. The main important aromatic and color compounds in R. damascena are 2-PE alcohol and anthocyanins, which are synthesized by PAR and ANS genes, respectively11. RhGT1 enzyme plays a role in anthocyanin biosynthesis by glycosylating precursors, which stabilizes the critical molecule21,104, that has an important function in the glycosylation of secondary metabolites and hormone regulation in plants. FLS is another key enzyme gene related to flower color105 and the biosynthesis pathway of flavonoids17. It catalyzes the production of flavonol from dihydro flavonol106 and plays a role in determining the synthesis of flavonol glycosides, which is one of the main branches of the flavonoid pathway107.

Different studies have been conducted on the expression of the ANS gene, which is a key gene in anthocyanin biosynthesis.Zhao et al. found that increasing ANS gene expression in Paeonia led to an increase in anthocyanin content in petals108, which is consistent with the results of the present study in ACC.1. Moreover, it was observed that there was no color change in Lisianthus plants due to the low expression of the ANS gene109, which is consistent with the results of the present study in ACC.26. Xu et al.110 confirmed that the reddening of leaves in red maple is regulated by the GT1 family genes, which affect anthocyanin accumulation. These findings are consistent with previous studies on the high expression of the FLS gene in white flowers of tobacco, which leads to the predominant accumulation of flavonol and a lack of anthocyanin111. Furthermore, Hu et al. studied three different colors of Scutellaria baicalensis petals (white, pink, and purple) and determined that the reduction in anthocyanin biosynthesis in white petals was due to the decreased activity of certain promoter regions of the ANS gene. This clarifies the mechanisms behind the color loss phenotype in the white flower of this plant112. Previous studies investigated molecular and phytochemical changes in four landraces of R. damascena in three growth stages and found that high expression of the RhANS gene, the key gene in anthocyanin biosynthesis, leads to an increase in anthocyanin pigments in petals. The highest amount of anthocyanin was observed in growth (stage b), while an increase in cell acidity in growth (stage c) led to a decrease in anthocyanin content11.

The clustering of network genes provides valuable insights into gene interactions, practical topics, and potential regulatory networks. Further investigation of these genes and their roles in specific pathways will enhance our understanding of cellular processes. The grouping of MYB1, ANS, GT1 and GGPPS genes into a single cluster could suggest analogous expression trends or operational correlations. The shared expression patterns could be attributed to similar regulatory mechanisms, overlapping biological pathways, or interactions within cellular processes. Studies have indicated that the interplay between the MYB1 and ANS genes might have a regulatory effect on the biosynthesis of anthocyanins in onions, thereby influencing the coloration of the onion tissue113. The GGPPS gene holds a crucial role in the carotenoid biosynthesis pathway. This gene instigates the production of ketonic compounds, specifically C13-norisoprenoids, which are a result of the CCD1 gene's activity. Interestingly, flowering plants determine the color or aroma of their flowers through mutual relationships among their biosynthetic pathways, aligning with the growth of the petals. In this study, a connection is observed between the CCD1 and GGPPS genes, placing them in the same group. The CCD1 gene is responsible for altering petal color and increasing the amount of carotenoid components. Similarly, the GGPPS gene could potentially be responsible for the production of volatile components and the development of carotenoid pigments. In multiple studies, shared genes that have experienced significant upregulation or downregulation have been used to explore biological, molecular, and cellular pathways. Broadly speaking, this collection of genes may influence color, aroma, and certain pathways that are instrumental in the operational process of this characteristic. The findings from these studies could enhance our comprehension of genetic control in plants and their stress reaction.

Conclusion

The findings of this research suggest that the MYB1 and CCD1 genes play an important role in determining the scent and color of rose flowers. The overexpression of the MYB1 transcription factor was found to significantly increase the expression of the ANS gene, indicating that MYB1 positively regulates ANS. Additionally, the application of the MeJA treatment was found to have a positive effect on the relative expression of most genes, except GGPPS. The study also showed that there were 26 different compounds in four samples using GC/MS analysis. The production of monoterpenes, sesquiterpenes, and aliphatic compounds in white flowers was found to increase after 48 h of MeJA application. In contrast, the application of MeJA to pink flowers led to a decrease in terpenoid compounds and an increase in aliphatic compounds. Further research is needed to fully understand the mechanisms behind the association between MeJA treatment and gene expression. The heatmap analysis revealed that the PS2 sample, representing the fully open flower stage of pink petals, is a superior specimen due to its substantial upregulation in the expression of genes GT1, GGPPS, MYB1, and ANS. While this study highlights the genetic potential of PS2 for enhanced essential oil production, future research should include detailed metabolite profiling, particularly focusing on citronellol, to fully validate PS2’s potential for the perfumery industry.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.