Flower Color Formation Research Articles

Molecular mechanism of flower colour formation in Rhododendron simsii Planchon revealed by integration of microRNAome and RNAomics.

Systems-wide understanding of gene expression profile regulating flower colour formation in Rhododendron simsii Planchon is insufficient. In this research, integration analysis of ribonucleic acid (RNA)omics and microRNAome were performed to reveal the molecular mechanism of flower colour formation in three R. simsii varieties with red, pink and crimson flowers, respectively. Totally, 3129, 5755 and 5295differentially expressed gene (DEG)s were identified through comparative transcriptome analysis between 'Red variety' and 'Pink variety' (1507 up-regulated and 1622 down-regulated), 'Red variety' and 'Crimson variety' (2148 up-regulated 3607 down-regulated), as well as 'Pink variety' and 'Crimson variety' (2089 up-regulated and 3206 down-regulated), which were involved in processes of 'catalytic activity', 'binding', 'metabolic process' and 'cellular process', as well as pathways of 'metabolic pathways', 'biosynthesis of secondary metabolites', 'plant-pathogen interaction' and 'phenylpropanoid biosynthesis'. A total of 215 miRNAs, containing 153 known miRNAs belonging to 57 families and 62 novel miRNA, were involved in flower colour formation. In particular, 55 miRNAs were significantly differently expressed. Based on miRNA-mRNA regulatory network, ath-miR5658 could affect the synthesis of pelargonidin, cyanidin and delphinidin through downregulating accumulation of anthocyanidin 3-O-glucosyltransferase; ath-miR868-3p could regulate isoflavonoid biosynthesis through downregulating expression of CYP81E1/E7; ath-miR156g regulated the expression of flavonoid 3',5'-hydroxylase; and ath-miR829-5p regulated flavonol synthasein flavonoid biosynthesis process. This research will provide important roles in breeding new varieties with rich flower colour.

Unbalanced Expression of Structural Genes in Carotenoid Pathway Contributes to the Flower Color Formation of the Osmanthus Cultivar 'Yanzhi Hong'.

Carotenoids are important natural pigments that are responsible for the fruit and flower colors of many plants. The composition and content of carotenoid can greatly influence the color phenotype of plants. However, the regulatory mechanism underling the divergent behaviors of carotenoid accumulation, especially in flower, remains unclear. In this study, a new cultivar Osmanthus fragrans 'Yanzhi Hong' was used to study the regulation of carotenoid pigmentation in flower. Liquid chromatograph-mass spectrometer (LC-MS) analysis showed that β-carotene, phytoene, lycopene, γ-carotene, and lutein were the top five pigments enriched in the petals of 'Yanzhi Hong'. Through transcriptome analysis, we found that the expression of the structural genes in carotenoid pathway was imbalanced: most of the structural genes responsible for lycopene biosynthesis were highly expressed throughout the flower developmental stages, while those for lycopene metabolism kept at a relatively lower level. The downregulation of LYCE, especially at the late developmental stages, suppressed the conversion from lycopene to α-carotene but promoted the accumulation of β-carotene, which had great effect on the carotenoid composition of 'Yanzhi Hong'. Ethylene response factor (ERF), WRKY, basic helix-loop-helix (bHLH), v-myb avian myeloblastosis viral oncogene homolog (MYB), N-Acetylcysteine (NAC), auxin response factor (ARF), and other transcription factors (TFs) have participated in the flower color regulation of 'Yanzhi Hong', which formed co-expression networks with the structural genes and functioned in multiple links of the carotenoid pathway. The results suggested that the cyclization of lycopene is a key link in determining flower color. The modification of the related TFs will break the expression balance between the upstream and downstream genes and greatly influence the carotenoid profile in flowers, which can be further used for creating colorful plant germplasms.

Multi-omics analysis revealed the difference in pigment accumulation profiles between Syringa oblata and S. oblata var. alba

Syringa oblata (So) and its variety S. oblata var. alba (Soa) are famous flowering shrubs with high ornamental value due to their beautiful inflorescence. The flower colors of So (purple) and Soa (white) differ significantly. In this study, pigment contents were detected and flavonoid and anthocyanin metabolites were determined by UPLC-ESI-MS/MS methods. Combined with transcriptome analysis, the key genes related to anthocyanin accumulation were obtained to explore the reasons for the difference in flower colors. The results showed that the total anthocyanin content was obviously higher in So than in Soa. The differential metabolites annotated in anthocyanin biosynthesis were significantly up-regulated, and conversely, the differential metabolites in biosynthesis of secondary metabolites and flavone and flavonol biosynthesis were down-regulated in So compared by Soa, with similar results revealed by the analysis of the top 20 fold change metabolites. It is explained that the anthocyanins and chlorophylls were the key pigment for the difference in flower color between So and Soa. Furthermore, the quantitative analysis of anthocyanins showed that delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside were the major anthocyanins in So, and their contents were significantly higher than other anthocyanins. However, the levels of major anthocyanins in Soa were very low, resulting in its white flower. In addition, high levels of kaempferol-3-O-rutinoside, rutin, quercetin-3-O-glucoside and naringenin were detected in flowers. These metabolites could play the roles of co-pigments in So and major pigments in Soa, respectively. Cyanidin-3-O-rutinoside were markedly elevated from initial flower bud (IFB) to flower bud (FB), which contributed to the anthocyanin accumulation and the flower color change in So, while the reduction of chlorophyll content was the main reason for the change in flower color from green to white in Soa. The integrated analysis showed that the genes (F3GT, FLS and F3H) might play the key roles in flower color formation. The study revealed the reason for the flower color variation between So and Soa and the flower color change during the development. It will provide the metabolites fundamental for research on flower color breeding in Syringa.

De novo transcriptome analysis identifies RpMYB1 as an activator of anthocyanin biosynthesis in Rehmannia piasezkii

Rehmannia piasezkii is a kind of medicinal plants, of the Orobanchaceae family, and well known for its large pink or purple corolla. However, no research on the molecular mechanism of flower color formation in R. piasezkii has been conducted so far. In this study, we investigated the transcriptome of root, stem, leaf and corollas of R. piasezkii using transcriptome sequencing technology and assembled 144,582 unigenes. A total of 58 anthocyanin biosynthetic genes were identified in the R. piasezkii transcriptome, fourteen of which were highly correlated with anthocyanin content, especially RpF3H2, RpDFR2, RpANS1, RpANS2 and RpUFGT. Totally, 35 MYB genes with FPKM values greater than 5 were identified in the R. piasezkii transcriptome, including an R2R3 MYB transcriptional factor RpMYB1, which belongs to subgroup 6 of the R2R3 MYB family. Agrobacterium-mediated transient expression of Nicotiana benthamiana revealed that overexpression of RpMYB1 could activate the expression of structural genes in anthocyanin synthesis pathway and promote the accumulation of anthocyanins in N. benthamiana leaves, indicating that RpMYB1 is a positive regulator of anthocyanin synthesis. Furthermore, combined transient overexpression of RpMYB1 with RpANS1, RpMYB1+RpANS1 with other structural genes all could further enhance the accumulation of anthocyanins in N. benthamiana leaves. Permanent overexpression of RpMYB1 in R. glutinosa promoted anthocyanin accumulation and expression levels of RgCHS, RgF3H, RgDFR and RgANS. Further evidence from dual-luciferase assay suggested that RpMYB1 could bind to the promoter of RpDFR2 and hence activating its expression. These findings provide insight into the molecular regulation in anthocyanin biosynthesis in R. piasezkii and provide valuable genetic resources for the genetic improvement of flower color.

An O-methyltransferase gene, RrCCoAOMT1, participates in the red flower color formation of Rosa rugosa

Rosa rugosa, a valuable ornamental plant species, has high aromatic, ornamental and medicinal values. Diversification of flower color is one of the main purposes for ornamental horticultural breeding, especially the red phenotype. Understanding the molecular mechanism of red color formation regulated by O-methyltransferase (OMT) during anthocyanin biosynthesis is necessary. In this study, 38 candidate members belonging to the CCoAOMT (11) and COMT (27) subfamilies were systematically identified with average lengths of 227 and 360 amino acids, respectively. Two subfamily members had different chromosome distribution characteristics, exon/intron structures, and conserved motifs, consistent with the phylogenetic analysis result of 257 OMT members from six species. Cis-elements in the promoter region of RrCCoAOMTs were mainly involved in hormone and stress response. RrCCoAOMT1 was mainly expressed in petals, and its expression level was significantly decreased in red flowers with high relative percentage of cyanidin. Moreover, RrCCoAOMT1 was separated from other RrCCoAOMTs based on the multiple sequence alignment analysis. Silent expression of RrCCoAOMT1 caused the formation of red/deep-pink phenotypes on purple- and pink-flowered germplasms with an increase in the ratio of Cy3G5G to Pn3G5G. Overall, RrCCoAOMT1 was involved in the modification of red petal color in R. rugosa via changing the ratio of Cy3G5G to Pn3G5G, and may be transcriptionally regulated by MYB transcription factors. These findings provided a theoretical foundation for further studies on the regulation mechanism of red phenotype formation in R. rugosa, and provided important gene resources for flower color improvement breeding.

Integration of Metabolomic and Transcriptomic Analyses Reveals the Molecular Mechanisms of Flower Color Formation in Prunus mume.

Flower color is an important trait that affects the economic value of Prunus mume, a famous ornamental plant in the Rosaceae family. P. mume with purple-red flowers is uniquely charming and highly favored in landscape applications. However, little is known about its flower coloring mechanism, which stands as a critical obstacle on the path to innovative breeding for P. mume flower color. In this study, transcriptomic and targeted metabolomic analyses of purple-red P. mume and white P. mume were performed to elucidate the mechanism of flower color formation. In addition, the expression patterns of key genes were analyzed using an RT-qPCR experiment. The results showed that the differential metabolites were significantly enriched in the flavonoid synthesis pathway. A total of 14 anthocyanins emerged as the pivotal metabolites responsible for the differences in flower color between the two P. mume cultivars, comprising seven cyanidin derivatives, five pelargonium derivatives, and two paeoniflorin derivatives. Moreover, the results clarified that the metabolic pathway determining flower color in purple-red P. mume encompasses two distinct branches: cyanidin and pelargonidin, excluding the delphinidin branch. Additionally, through the integrated analysis of transcriptomic and metabolomic data, we identified 18 key genes responsible for anthocyanin regulation, thereby constructing the gene regulatory network for P. mume anthocyanin synthesis. Among them, ten genes (PmCHI, PmGT2, PmGT5, PmGST3, PmMYB17, PmMYB22, PmMYB23, PmbHLH4, PmbHLH10, and PmbHLH20) related to anthocyanin synthesis were significantly positively correlated with anthocyanin contents, indicating that they may be the key contributors to anthocyanin accumulation. Our investigation contributes a novel perspective to understanding the mechanisms responsible for flower color formation in P. mume. The findings of this study introduce novel strategies for molecular design breeding aimed at manipulating flower color in P. mume.

Genome assembly and multi-omic analyses reveal the mechanisms underlying flower color formation in Torenia fournieri.

Torenia fournieri Lind. is an ornamental plant that is popular for its numerous flowers and variety of colors. However, its genomic evolutionary history and the genetic and metabolic bases of flower color formation remain poorly understood. Here, we report the first T. fournieri reference genome, which was resolved to the chromosome scale and was 164.4 Mb in size. Phylogenetic analyses clarified relationships with other plant species, and a comparative genomic analysis indicated that the shared ancestor of T. fournieri and Antirrhinum majus underwent a whole genome duplication event. Joint transcriptomic and metabolomic analyses identified many metabolites related to pelargonidin, peonidin, and naringenin production in rose (TfR)-colored flowers. Samples with blue (TfB) and deep blue (TfD) colors contained numerous derivatives of petunidin, cyanidin, quercetin, and malvidin; differences in the abundances of these metabolites and expression levels of the associated genes were hypothesized to be responsible for variety-specific differences in flower color. Furthermore, the genes encoding flavonoid 3-hydroxylase, anthocyanin synthase, and anthocyanin reductase were differentially expressed between flowers of different colors. Overall, we successfully identified key genes and metabolites involved in T. fournieri flower color formation. The data provided by the chromosome-scale genome assembly establish a basis for understanding the differentiation of this species and will facilitate future genetic studies and genomic-assisted breeding.

Selection and Validation of qRT-PCR Internal Reference Genes to Study Flower Color Formation in Camellia impressinervis.

Real-time quantitative PCR (qRT-PCR) is a pivotal technique for gene expression analysis. To ensure reliable and accurate results, the internal reference genes must exhibit stable expression across varied experimental conditions. Currently, no internal reference genes for Camellia impressinervis have been established. This study aimed to identify stable internal reference genes from eight candidates derived from different developmental stages of C. impressinervis flowers. We employed geNorm, NormFinder, and BestKeeper to evaluate the expression stability of these candidates, which was followed by a comprehensive stability analysis. The results indicated that CiTUB, a tubulin gene, exhibited the most stable expression among the eight reference gene candidates in the petals. Subsequently, CiTUB was utilized as an internal reference for the qRT-PCR analysis of six genes implicated in the petal pigment synthesis pathway of C. impressinervis. The qRT-PCR results were corroborated by transcriptome sequencing data, affirming the stability and suitability of CiTUB as a reference gene. This study marks the first identification of stable internal reference genes within the entire genome of C. impressinervis, establishing a foundation for future gene expression and functional studies. Identifying such stable reference genes is crucial for advancing molecular research on C. impressinervis.

Integrated Metabolomic and Transcriptomic Profiles Provide Insights into the Mechanisms of Anthocyanin and Carotenoid Biosynthesis in Petals of Medicago sativa ssp. sativa and Medicago sativa ssp. falcata.

The petals of Medicago sativa ssp. sativa and M. sativa ssp. falcata are purple and yellow, respectively. Free hybridization between M. sativa ssp. sativa and M. sativa ssp. falcata has created hybrids with various flower colors in nature. Moreover, the flower colors of alfalfa are closely correlated with yield, nutritional quality, stress tolerance and other agronomic characteristics. To elucidate the underlying mechanisms of flower color formation in M. sativa ssp. sativa and M. sativa ssp. falcata, we conducted an integrative analysis of the transcriptome and metabolome of alfalfa with three different petal colors (purple, yellow and cream). The metabolic profiles suggested that anthocyanins and carotenoids are the crucial pigments in purple and yellow flowers, respectively. A quantitative exploration of the anthocyanin and carotenoid components indicated that the accumulations of cyanidin, delphinidin, peonidin, malvidin, pelargonidin and petunidin derivatives are significantly higher in purple flowers than in cream flowers. In addition, the content of carotenes (phytoene, α-carotene and β-carotene) and xanthophylls (α-cryptoxanthin, lutein, β-cryptoxanthin, zeaxanthin, antheraxanthin and violaxanthin derivatives) was markedly higher in yellow flowers than in cream flowers. Furthermore, we found that delphinidin-3,5-O-diglucoside and lutein were the predominant pigments accumulated in purple and yellow flowers, respectively. The transcriptomic results revealed that twenty-five upregulated structural genes (one C4H, three 4CL, twelve CHS, two CHI, one F3H, one F3'H, one F3'5'H and four DFR) are involved in the accumulation of anthocyanins in purple flowers, and nine structural genes (two PSY, one ZDS, two CRTISO, two BCH, one ZEP and one ECH) exert an effect on the carotenoid biosynthesis pathway in yellow flowers. The findings of this study reveal the underlying mechanisms of anthocyanin and carotenoid biosynthesis in alfalfa with three classic flower colors.

Genome-Wide Identification and Expression Analysis of the MYB Transcription Factor Family in Salvia nemorosa.

The MYB transcription factor gene family is among the most extensive superfamilies of transcription factors in plants and is involved in various essential functions, such as plant growth, defense, and pigment formation. Salvia nemorosa is a perennial herb belonging to the Lamiaceae family, and S. nemorosa has various colors and high ornamental value. However, there is little known about its genome-wide MYB gene family and response to flower color formation. In this study, 142 SnMYB genes (MYB genes of S. nemorosa) were totally identified, and phylogenetic relationships, conserved motifs, gene structures, and expression profiles during flower development stages were analyzed. A phylogenetic analysis indicated that MYB proteins in S. nemorosa could be categorized into 24 subgroups, as supported by the conserved motif compositions and gene structures. Furthermore, according to their similarity with AtMYB genes associated with the control of anthocyanin production, ten SnMYB genes related to anthocyanin biosynthesis were speculated and chosen for further qRT-PCR analyses. The results indicated that five SnMYB genes (SnMYB75, SnMYB90, SnMYB6, SnMYB82, and SnMYB12) were expressed significantly differently in flower development stages. In conclusion, our study establishes the groundwork for understanding the anthocyanin biosynthesis of the SnMYB gene family and has the potential to enhance the breeding of S. nemorosa.

The formation and evolution of flower coloration in Brassica crops.

The flower coloration of Brassica crops possesses significant application and economic value, making it a research hotspot in the field of genetics and breeding. In recent years, great progress has been made in the research on color variation and creation of Brassica crops. However, the underlying molecular mechanisms and evolutional processes of flower colors are poorly understood. In this paper, we present a comprehensive overview of the mechanism of flower color formation in plants, emphasizing the molecular basis and regulation mechanism of flavonoids and carotenoids. By summarizing the recent advances on the genetic mechanism of flower color formation and regulation in Brassica crops, it is clearly found that carotenoids and anthocyanins are major pigments for flower color diversity of Brassica crops. Meantime, we also explore the relationship between the emergence of white flowers and the genetic evolution of Brassica chromosomes, and analyze the innovation and multiple utilization of Brassica crops with colorful flowers. This review aims to provide theoretical support for genetic improvements in flower color, enhancing the economic value and aesthetic appeal of Brassica crops.

Transcriptome analysis of genes involved in flower and leaf color of Oncidium by RNA-seq

Oncidium, an important tropical orchid, has high ornamental value due to its specific color and occupies a significant market position for the worldwide flower. Transcriptome analysis of flower and leaf color formation provides new sources for producing novel Oncidium hybridum cultivars. We sequenced 12 samples of flowers (yellow and white) and leaves (striped and regular) of O. hybridum and assembled 381,136 and 453,566 unigene sequences from RNA-seq data, respectively. Among unigenes, 662 and 1,324 differentially expressed genes were identified in flower and leaf samples, respectively. Gene ontology and pathway enrichment showed that secondary metabolite biosynthetic pathways were responsible for flower and leaf color formation. It was determined that UGT75C1, E2.4.1.115, CCD7, E2.1.1.76, and CCoAOMT are involved in regulating flower color, and UGT75C1, LHCB, UGT, RP-L18Ae, and ABCB1 play crucial roles in regulating leaf color. Kyoto Encyclopedia of Genes and Genomes analysis revealed that UGT75C1 was significantly enriched in the anthocyanin biosynthetic pathway, showing effects on flower and leaf colors. This study was the first detailed analysis of the molecular mechanisms of O. hybridum flower and leaf colors, and the results advanced the understanding of the genetic basis of flower and leaf colors; they also provided additional support for improving commercial value and producing novel cultivars of O. hybridum.

Molecular mechanism of different flower color formation of Cymbidium ensifolium.

Cymbidium ensifolium is one of the national orchids in China, which has high ornamental value with changeable flower colors. To understand the formation mechanism of different flower colors of C. ensifolium, this research conducted transcriptome and metabolome analyses on four different colored sepals of C. ensifolium. Metabolome analysis detected 204 flavonoid metabolites, including 17 polyphenols, 27 anthocyanins, 75 flavones, 34 flavonols, 25 flavonoids, 18 flavanones, and 8 isoflavones. Among them, purple-red and red sepals contain a lot of anthocyanins, including cyanidin, pelargonin, and paeoniflorin, while yellow-green and white sepals have less anthocyanins detected, and their metabolites are mainly flavonols, flavanones and flavonoids. Transcriptome sequencing analysis showed that the expression levels of the anthocyanin biosynthetic enzyme genes in red and purple-red sepals were significantly higher than those in white and yellow-green sepals of C. ensifolium. The experimental results showed that CeF3'H2, CeDFR, CeANS, CeF3H and CeUFGT1 may be the key genes involved in anthocyanin production in C. ensifolium sepals, and CeMYB104 has been proved to play an important role in the flower color formation of C. ensifolium. The results of transformation showed that the CeMYB104 is involved in the synthesis of anthocyanins and can form a purple-red color in the white perianth of Phalaenopsis. These findings provide a theoretical reference to understand the formation mechanism of flower color in C. ensifolium.

Multi-omics analysis reveals new insights into the bicolor flower phenotype of Paeonia suffruticosa ‘Erqiao’

The bicolor phenotype is conducive to revealing the changes in plant flower color and an important scientific model for studying the regulatory mechanism of flower color. The flowers of Paeonia suffruticosa ‘Erqiao’ have two colors, pink-white and purple-red, and bicolor flowers are rare, possessing unique ornamental value. The distribution of the two colors in a bicolor ‘Erqiao’ flower is similar to a pie chart and can be divided into two distinct parts, with the percentages of the two parts varying. We analyzed the mechanism of flower color formation in ‘Erqiao’ using transcriptomics, proteomics, and metabolomics. The results showed that the differentially abundant metabolites (DAMs), differentially abundant proteins (DAPs), and differentially expressed genes (DEGs) in the two petal types were primarily enriched in phenylpropanoid and flavonoid biosynthesis. Compared with purple-red petals, the PsF3′H protein content in pink-white petals was lower than that in purple-red petals. The content of PsF3′H’s downstream metabolites decreased, while that of some metabolites in the collateral branch of phenylpropanoid biosynthesis increased. The PsF3′H sequence analysis of different P. suffruticosa cultivars and ‘Erqiao’ tissues revealed that the 6 bp insertion mutation of PsF3′H existed only in ‘Erqiao’ and was stably expressed in the pink-white petals. Using virus-induced gene silencing to silence PsF3′H in ‘Erqiao’ petals, the color of the petals faded significantly. Therefore, through multi-omics analysis, we found that the insertion mutation of PsF3′H reduced the synthesis of the PsF3′H protein and led to the obstruction of anthocyanin synthesis; furthermore, other branches and metabolic pathways of the upstream pathway were more active, thus changing the synthesis direction of the entire metabolic pathway. The 6-bp insertion mutation of PsF3′H may be a reason for the rare appearance of bicolor flowers and the unstable ratio of two-colored petals in ‘Erqiao’. This study provides new insights into the formation of the bicolor flower phenotype in ‘Erqiao’.

Exploring the molecular mechanism of coloration differences in two Meconopsis wilsonii subspecies: australis and orientalis

Flower color diversity is a key taxonomic trait in Meconopsis species, enhancing their appeal as ornamental flowers. However, knowledge of the molecular mechanisms of flower color formation in Meconopsis species is still limited. M. wilsonii subsp. australis (Australis) and M. wilsonii subsp. orientalis (Orientalis) have a developmental stage presenting red-purple flowers, while Orientalis also presents blue coloration at the full-bloom period, making them an important model for exploring the mechanism of blue flower formation in M. wilsonii. In this study, we collected petals from Australis and Orientalis at different developmental stages to compare the coloration differences between the two species and detect the molecular mechanisms of blue color in Orientalis. We identified that cyanidin was the main anthocyanin in the flowers of both species, and the blue color in Orientalis primarily arises from anthocyanins (Cyanidin-3-O-sambubioside). RNA sequencing analysis was performed to detect the gene expression in the anthocyanin biosynthesis pathway, and the results suggested that gene regulation for anthocyanin biosynthesis may not be the direct reason for blue color formation in Orientalis. In addition, the growth solid of Orientalis was rich in Fe and Mg ions, and a large amount of Fe and Mg ions accumulated in the petals of Orientalis. Combined with the gene functional enrichment results, we found that the purple and red-purple colors of these two species were presented by different glycosylation levels of cyanidin, while the violet color of Orientalis might be the results of metalloanthocyanins by Fe and Mg ions, which also relieved the toxicity caused by the high content of Fe and Mg ions in its cells. The environmental adaptation-related genes were highly expressed of in both species, such as adaptation to desiccation, water deprivation, freezing, etc. Our results revealed the coloration differences between Australis and Orientalis and described the molecular mechanisms of blue coloration in Orientalis. The data in our analysis could enrich the genetic resources for M. wilsonii for further studies.

Long non-coding RNA-mediated competing endogenous RNA regulatory network during flower development and color formation in Melastoma candidum.

M. candidum, an evergreen shrubby flower known for its superior adaptation ability in South China, has gained increased attention in garden applications. However, scant attention has been paid to its flower development and color formation process at the non-coding RNA level. To fill this gap, we conducted a comprehensive analysis based on long non-coding RNA sequencing (lncRNA-seq), RNA-seq, small RNA sequencing (sRNA-seq), and widely targeted metabolome detection of three different flower developmental stages of M. candidum. After differentially expressed lncRNAs (DElncRNAs), differentially expressed mRNAs (DEmRNAs), differentially expressed microRNAs (DEmiRNAs), and differentially synthesized metabolites (DSmets) analyses between the different flower developmental stages, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted to identify some key genes and metabolites in flavonoid, flavone, anthocyanin, carotenoid, and alkaloid-related GO terms and biosynthetic pathways. Three direct-acting models, including antisense-acting, cis-acting, and trans-acting between lncRNAs and mRNAs, were detected to illustrate the direct function of lncRNAs on target genes during flower development and color formation. Based on the competitive endogenous RNA (ceRNA) regulatory theory, we constructed a lncRNA-mediated regulatory network composed of DElncRNAs, DEmiRNAs, DEmRNAs, and DSmets to elucidate the indirect role of lncRNAs in the flower development and color formation of M. candidum. By utilizing correlation analyses between DERNAs and DSmets within the ceRNA regulatory network, alongside verification trials of the ceRNA regulatory mechanism, the study successfully illustrated the significance of lncRNAs in flower development and color formation process. This research provides a foundation for improving and regulating flower color at the lncRNA level in M. candidum, and sheds light on the potential applications of non-coding RNA in studies of flower development.

CmMYB3-like negatively regulates anthocyanin biosynthesis and flower color formation during the post-flowering stage in Chrysanthemum morifolium

Color fading caused by a decrease in anthocyanin accumulation during the post-flowering stage significantly affects postharvest quality of chrysanthemum. However, the underlying mechanism by which anthocyanin accumulation decreases during the post-flowering stage still unclear, which greatly restricts design of molecular breeding in chrysanthemum. Here, a chrysanthemum SG7 R2R3 MYB transcription factor (TF), CmMYB3-like, was identified to have a function in regulating anthocyanin biosynthesis during the post-flowering stage. Quantitative real time PCR (qRT-PCR) assays showed that the expression of CmMYB3-like was gradually downregulated when anthocyanin content increased during the flowering stage and was significantly upregulated during the post-flowering stage. Genetic transformation of chrysanthemum and dual-luciferase assays in N. benthamiana leaves showed that CmMYB3-like suppressed anthocyanin accumulation by inhibiting the transcription of CmCHS and CmANS directly and that of CmF3H indirectly. However, overexpression or suppression of CmMYB3-like did not affect the biosynthesis of flavones or flavonols. Genetic transformation of chrysanthemum revealed that the overexpression of CmMYB3-like inhibited anthocyanin accumulation, but its suppression prevented the decrease in anthocyanin accumulation during the post-flowering stage. Our results revealed a crucial role of CmMYB3-like in regulating the color of petals during the post-flowering stage and provided a target gene for molecular design breeding to improve the postharvest quality of chrysanthemum.

Transcriptomic Analysis Reveals the Regulatory Mechanism of Color Diversity in Rhododendron pulchrum Sweet (Ericaceae).

Rhododendron pulchrum Sweet is a traditional ornamental plant cultivated in China and presents a great variation in petal coloration. However, few studies have been performed to reveal the genes involved and the regulatory mechanism of flower color formation in this plant. In this study, to explore the underlying genetic basis of flower color formation, transcriptome analysis was performed by high-throughput sequencing techniques on four petal samples of different colors: purple, pink, light pink, and white. Results show that a total of 35.55 to 40.56 million high-quality clean reads were obtained, of which 28.56 to 32.65 million reads were mapped to the reference genome. For their annotation, 28,273, 18,054, 24,301, 19,099, and 11,507 genes were allocated to Nr, Swiss-Prot, Pfam, GO, and KEGG databases, correspondingly. There were differentially expressed genes among the four different petal samples, including signal-transduction-related genes, anthocyanin biosynthesis genes, and transcription factors. We found that the higher expressed levels of genes associated with flavonol synthase (FLS) might be the key to white formation, and the formation of red color may be related to the higher expression of flavanone 4-reductase (DFR) families. Overall, our study provides some valuable information for exploring and understanding the flower color intensity variation in R. pulchrum.

Transcriptome analysis of flower colour reveals the correlation between SNP and differential expression genes in Phalaenopsis.

Phalaenopsis is an important ornamental plant that has great economic value in the world flower market as one of the most popular flower resources. To investigate the flower colour formation of Phalaenopsis at the transcription level, the genes involved in flower color formation were identified from RNA-seq in this study. In this study, white and purple petals of Phalaenopsis were collected and analyzed to obtained (1) differential expression genes (DEGs) between white and purple flower color and (2) the association between single nucleotide polymorphisms (SNP) mutations and DEGs at the transcriptome level. The results indicated that a total of 1,175 DEGs were identified, and 718 and 457 of them were up- and down-regulated genes, respectively. Gene Ontology and pathway enrichment showed that the biosynthesis of the secondary metabolites pathway was key to color formation, and the expression of 12 crucial genes (C4H, CCoAOMT, F3'H, UA3'5'GT, PAL, 4CL, CCR, CAD, CALDH, bglx, SGTase, and E1.11.17) that are involved in the regulation of flower color in Phalaenopsis. This study reported the association between the SNP mutations and DEGs for color formation at RNA level, and provides a new insight to further investigate the gene expression and its relationship with genetic variants from RNA-seq data in other species.

CircRNA-mediated ceRNA regulatory network during flower development and color formation in Melastoma candidum

Melastoma candidum D. Don is widely distributed in South China. However, its application in the garden industry is not ideal due to its sole flower color; thus, its color needs urgent improvement. In this study, we conducted high throughput transcriptome sequencing for circular RNA (circRNA), mRNA, small RNA (sRNA), and widely targeted metabolome in flowers at three developmental stages. RNA expression and metabolite abundance analyses identified differentially expressed circRNAs (DEcircRNAs), mRNAs (DEmRNAs), miRNAs (DEmiRNAs), and differentially synthesized metabolites (DSmets). Alternative variants of genes were given statistics and the reason for their occurrence was analyzed. GO enrichment and KEGG analyses were conducted on the source gene of DEcircRNAs and KEGG analyses on DSmets. Key genes and metabolites were identified in flavonoid, flavone, anthocyanin, carotenoid, and alkaloid biosynthetic pathways. Based on the above analyses, we built a circRNA-mediated ceRNA regulatory network. Nine circRNA-mediated ceRNA regulatory routines were obtained. Within the ceRNA regulatory network, circRNAs and mRNAs displayed positive correlations, both of which exhibit negative correlations with miRNAs, indicating their important roles during flower development and color formation of M. candidum. Amongst, circ_000434-miR5207-McF3H could regulate the biosynthesis of Dihydroquercetin, Dihydromyricetin, and Pinobanksin; circ_000302-miR319–5p-McHST and circ_00012 -miR826-McTAT could regulate the biosynthesis of p-Counmaryl shikimic acid. Our research explored the role of circRNAs in flower development and the color formation process and provided a theoretical foundation for flower color improvement in M. candidum.