Narcissus pseudonarcissus is an important bulbous plant with white or yellow perianths and light yellow to orange-red coronas, but little is known regarding the biochemical and molecular basis related to flower color polymorphisms. To investigate the mechanism of color formation, RNA-Seq of flower of two widely cultured cultivars (‘Slim Whitman’ and ‘Pinza’) with different flower color was performed. A total of 84,463 unigenes were generated from the perianths and coronas. By parallel metabolomic and transcriptomic analyses, we provide an overview of carotenoid biosynthesis, degradation, and accumulation in N. pseudonarcissus. The results showed that the content of carotenoids in the corona was higher than that in the perianth in both cultivars. Accordingly, phytoene synthase (PSY) transcripts have a higher abundance in the coronas than that in perianths. While the expression levels of carotenoid biosynthetic genes, like GGPPS, PSY, and LCY-e, were not significantly different between two cultivars. In contrast, the carotenoid degradation gene NpCCD4 was highly expressed in white-perianth cultivars, but was hardly detected in yellow-perianth cultivars. Silencing of NpCCD4 resulted in a significant increase in carotenoid accumulation, especially in all-trans-β-carotene. Therefore, we presume that NpCCD4 is a crucial factor that causes the low carotenoid content and color fading phenomenon of ‘Slim Whitman’ by mediating carotenoid turnover. Our findings provide mass RNA-seq data and new insights into carotenoid metabolism in N. pseudonarcissus.

Freesia hybrida is rich in flower colors with beautiful flower shapes and pleasant aroma. Flavonoids are vital to the color formation of its flowers. In this study, five Freesia cultivars with different flower colors were used to study on the level of accumulation of their flavonoids and expression of flavonoid-related genes and further explore new novel transcription factor (TF). Ultra-high-performance liquid chromatography and VION ion mobility quadrupole time-of-flight mass spectrometer (UPLC-Q-TOF-MS) were used to determine the flavonoids. Combined with transcriptome sequencing technology, the molecular mechanism of the flavonoid metabolism difference in Freesia was revealed. A total of 10 anthoxanthin components and 12 anthocyanin components were detected using UPLC-Q-TOF-MS. All six common anthocyanin aglycones in high plants, including cyanidin, delphinidin, petunidin, peonidin, malvidin, and pelargonidin, were detected in Freesia at first time in this study. In orange, yellow, and white cultivars, anthoxanthins gradually decreased with the opening of the petals, while in red and purple cultivars, anthoxanthins first increased and then decreased. No anthocyanin was detected in yellow and white cultivars, while anthocyanins increased with the opening of the petals and reached their maximum at the flowering stage (S3) in other three cultivars. The correlation analysis revealed that the color of Freesia petals was closely related to the composition and content of anthoxanthins and anthocyanins. Petals of five cultivars at S3 were then selected for transcriptome sequencing by using the Illumina Hiseq 4000 platform, and a total of 100,539 unigenes were obtained. There were totally 5,162 differentially expressed genes (DEGs) when the four colored cultivars were compared with the white cultivar at S3. Comparing all DEGs with gene ontology (GO), KEGG, and Pfam databases, it was found that the genes involved in the flavonoid biosynthesis pathway were significantly different. In addition, AP2, WRKY, and bHLH TF families ranked the top three among all differently expressed TFs in all DEGs. Quantitative real-time PCR (qRT-PCR) technology was used to analyze the expression patterns of the structural genes of flavonoid biosynthesis pathway in Freesia. The results showed that metabolic process was affected significantly by structural genes in this pathway, such as CHS1, CHI2, DFR1, ANS1, 3GT1, and FLS1. Cluster analysis was performed by using all annotated WRKY and AP2 TFs and the above structural genes based on their relatively expression. Four novel candidate TFs of WRKY and AP2 family were screened. Their spatiotemporal expression patterns revealed that these four novel TFs may participate in the regulation of the flavonoid biosynthesis, thus controlling its color formation in Freesia petals.

Tulipa gesneriana has rich cultivars with abundant flower colors; among them, black tulips have become precious cultivars for their rareness in nature. It is of great significance to understand its color formation mechanism for breeding new cultivars with a blackish flower color in the future. In this present study, petals at five developmental stages of Tulipa gesneriana ‘Queen of Night’ (shorted for ‘QN’), a typical black tulip cultivar, were collected to identity the flavonoid composition and compare the accumulation along with the flower development process, aiming to explore the metabolic mechanism of its flower coloration. By using UPLC-Q-TOF-MS, three anthocyanins and nineteen anthoxanthins (including eighteen flavonols and one flavone) were detected in the petals of ‘QN’. The anthocyanins were identified as delphinidin 3-o-rutinoside, cyanidin 3-o-rutinoside and pelargonidin 3-o-rutinoside, respectively. The main flavonols were identified as quercetin, kaempferol, isorhamnetin, naringin and their glycosides. The only one flavone substance was identified as an apigenin derivative. By comparing the content, anthocyanins were the most abundant substance in the petals of ‘QN’, and showed obvious regularity in the development process. With the flower opening, the anthocyanin content accumulated continuously, and reached the highest level at 3575.9 μg·g⁻¹·FW at S5, accounting for 80% of the total flavonoids. Among them, delphinidin 3-o-rutinoside and cyanidin 3-o-rutinoside made the main contribution to the coloration of ‘QN’. The content of anthoxanthins increased first and then decreased, reaching the highest to 1114.8 μg·g⁻¹·FW at S3. Quercetin and its glycosides were the most important flavonol substances in the petals of ‘QN’, accounting for more than 60% of the total anthoxanthins in the five stages. The content of a flavone identified was extremely low, suggesting a limited role in the flower color of ‘QN’. Taken together, the flower color presentation of ‘QN’ was closely related to the composition and accumulation of anthocyanins and flavonols; anthocyanins were the main substances that determine the petal coloration of ‘QN’, and the high content of flavonols played a role of co-pigmentation with these anthocyanins.

Freesia hybrida is a group of cultivars in the genus Freesia with a strong floral scent composed of diverse volatile organic compounds (VOCs). In this study, the VOCs of 34 F. hybrida were extracted and analyzed by headspace solid phase microextraction and gas chromatography mass spectrometry (HS-SPME-GC-MS). A total of 164 VOCs whose relative contents were higher than 0.05% were detected. The numbers of VOCs in all germplasms differed between 11 to 38, and the relative contents ranged from 32.39% to 94.28%, in which most germplasms were higher than 80%. Terpenoids, especially monoterpenes, were the crucial type of VOCs in most germplasms, of which linalool and D-limonene were the most frequently occurring. Principal component analysis (PCA) clearly separated samples based on whether linalool was the main component, and hierarchical clustering analysis (HCA) clustered samples into 4 groups according to the preponderant compounds linalool and (E)-β-ocimene. Comparison of parental species and hybrids showed heterosis in three hybrids, and the inherited and novel substances suggested that monoterpene played an important role in F. hybrida floral scent. This study established a foundation for the evaluation of Freesia genetic resources, breeding for the floral aroma and promoting commercial application.