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Background Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. However, in Chinese Narcissus ( Narcissus tazetta L. var. chinensis ), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear. Results In this work, genes related to the regulatory complex, NtbHLH1 and a R2R3-MYB NtMYB6, were cloned from Chinese Narcissus. Phylogenetic analysis indicated that NtbHLH1 belongs to the JAF13 clade of bHLH IIIf subgroup, while NtMYB6 was highly homologous to positive regulators of proanthocyanidin biosynthesis. Both NtbHLH1 and NtMYB6 have highest expression levels in basal plates of Narcissus, where there is an accumulation of proanthocyanidin. Ectopic over expression of NtbHLH1 in tobacco resulted in an increase in anthocyanin accumulation in flowers, and an up-regulation of expression of the endogenous tobacco bHLH AN1 and flavonoid biosynthesis genes. In contrast, the expression level of LAR gene was significantly increased in NtMYB6 -transgenic tobacco. Dual luciferase assays showed that co-infiltration of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese Narcissus DFR gene. Furthermore, a yeast two-hybrid assay confirmed that NtbHLH1 interacts with NtMYB6. Conclusions Our results suggest that NtbHLH1 may function as a regulatory partner by interacting directly with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.

R2R3-MYB transcription factors play important roles in the regulation of plant flavonoid metabolites. In the current study, NtMYB3, a novel R2R3-MYB transcriptional factor isolated from Chinese narcissus (Narcissus tazetta L. var. chinensis), was functionally characterized. Phylogenetic analysis indicated that NtMYB3 belongs to the AtMYB4-like clade, which includes repressor MYBs involved in the regulation of flavonoid biosynthesis. Transient assays showed that NtMYB3 significantly reduced red pigmentation induced by the potato anthocyanin activator StMYB-AN1 in agro-infiltrated leaves of tobacco. Over-expression of NtMYB3 decreased the red color of transgenic tobacco flowers, with qRT-PCR analysis showing that NtMYB3 repressed the expression levels of genes involved in anthocyanin and flavonol biosynthesis. However, the proanthocyanin content in flowers of transgenic tobacco increased as compared to wild type. NtMYB3 showed expression in all examined narcissus tissues; the expression level in basal plates of the bulb was highest. A 968 bp promoter fragment of narcissus FLS (NtFLS) was cloned, and transient expression and dual luciferase assays showed NtMYB3 repressed the promoter activity. These results reveal that NtMYB3 is involved in the regulation of flavonoid biosynthesis in narcissus by repressing the biosynthesis of flavonols, and this leads to proanthocyanin accumulation in the basal plate of narcissus.

R2R3 MYB transcription factors play key functions in the regulation of secondary metabolites. In the present study, a R2R3 MYB transcriptional factor NtMYB2 was identified from Chinese narcissus (Narcissus tazetta L. var. Chinensis Roem) and functionally characterized. NtMYB2 belongs to subgroup 4 of the R2R3 MYB transcription factor family that are related to repressor MYBs involved in the regulation of anthocyanin and flavonoids. Transient expression confirmed that NtMYB2 strongly reduced the red pigmentation induced by MYB- anthocyanin activators in agro-infiltrated tobacco leaves. Ectopic expression of NtMYB2 in tobacco significantly reduced the pigmentation and altered the floral phenotypes in transgenic tobacco flowers. Gene expression analysis suggested that NtMYB2 repressed the transcript levels of structural genes involved in anthocyanin biosynthesis pathway, especially the UFGT gene. NtMYB2 gene is expressed in all examined narcissus tissues; the levels of transcription in petals and corona is higher than other tissues and the transcription level at the bud stage was highest. These results show that NtMYB2 is involved in the regulation of anthocyanin biosynthesis pathway and may act as a repressor by down regulating the transcripts of key enzyme genes in Chinese narcissus.

Viromes of Chinese narcissus flowers were explored using transcriptome data from 20 samples collected at different flower development stages. Quality controlled raw data underwent de novo assembly, resulting in 5893 viral contigs that matched the seven virus species. The most abundant viruses were narcissus common latent virus (NCLV), narcissus yellow stripe virus (NYSV), and narcissus mottling-associated virus (NMaV). As flower development stages advanced, white tepal plants showed an increase in the proportion of viral reads, while the variation in viral proportion among yellow tepal plants was relatively small. Narcissus degeneration virus (NDV) dominated the white tepal samples, whereas NDV and NYSV prevailed in the yellow tepal samples. Potyviruses, particularly NDV, are the primary infectious viruses. De novo assembly generated viral contigs for five viruses, yielding complete genomes for NCLV, NDV, narcissus late season yellow virus (NLSYV), and NYSV. Phylogenetic analysis revealed genetic diversity, with distinct NCLV, NMaV, NDV, NLSYV, and NYSV groups. This study provides valuable insights into the viromes and genetic diversity of viruses in Chinese narcissus flowers.

Chinese narcissus (Narcissus tazetta var. chinensis) is one of the ten traditional flowers in China and a famous bulb flower in the world flower market. However, only white color tepals are formed in mature flowers of the cultivated varieties, which constrains their applicable occasions. Unfortunately, for lack of genome information of narcissus species, the explanation of tepal color formation of Chinese narcissus is still not clear. Concerning no genome information, the application of transcriptome profile to dissect biological phenomena in plants was reported to be effective. As known, pigments are metabolites of related metabolic pathways, which dominantly decide flower color. In this study, transcriptome profile and pigment metabolite analysis methods were used in the most widely cultivated Chinese narcissus “Jinzhanyintai” to discover the structure of pigment metabolic pathways and their contributions to white tepal color formation during flower development and pigmentation processes. By using comparative KEGG pathway enrichment analysis, three pathways related to flavonoid, carotenoid and chlorophyll pigment metabolism showed significant variations. The structure of flavonoids metabolic pathway was depicted, but, due to the lack of F3ʹ5ʹH gene; the decreased expression of C4H, CHS and ANS genes; and the high expression of FLS gene, the effect of this pathway to synthesize functional anthocyanins in tepals was weak. Similarly, the expression of DXS, MCT and PSY genes in carotenoids synthesis sub-pathway was decreased, while CCD1/CCD4 genes in carotenoids degradation sub-pathway was increased; therefore, the effect of carotenoids metabolic pathway to synthesize adequate color pigments in tepals is restricted. Interestingly, genes in chlorophyll synthesis sub-pathway displayed uniform down-regulated expression, while genes in heme formation and chlorophyll breakdown sub-pathways displayed up-regulated expression, which also indicates negative regulation of chlorophyll formation. Further, content change trends of various color metabolites detected by HPLC in tepals are consistent with the additive gene expression patterns in each pathway. Therefore, all three pathways exhibit negative control of color pigments synthesis in tepals, finally resulting in the formation of white tepals. Interestingly, the content of chlorophyll was more than 10-fold higher than flavonoids and carotenoids metabolites, which indicates that chlorophyll metabolic pathway may play the major role in deciding tepal color formation of Chinese narcissus.

The volatile components in single-flowered and double-flowered Chinese narcissus were identified by headspace-solid phase microextraction (HS-SPME) coupled with GC and GC/MS. Changes in aroma during the vase-life (days 0, 1, 2, 3, 4, 5 and 6) of two samples were also studied. A total of 35 compounds were identified, of which all were present in single-flowered and 26 in double-flowered samples. The main aroma components were (E)-β-ocimene, and benzyl acetate. Single-flowered narcissus have a higher percentage of benzyl acetate, while double-flowered narcissus have a higher percentage of 1,8-cineole. In vase-life, the total volatile component content peaked on day 2 for single-flowered and day 3 for the double-flowered narcissus. For both single-flowered and double-flowered narcissus flowers, the total content of volatile components had decreased significantly by day 4.

Chinese narcissus (Narcissus tazetta L. var. chinensis Roem.) is a popular flower in Asia. However, flower colors are limited with all cultivars having a white perianth and yellow corona. Previous studies have shown no anthocyanin accumulation in this species. The reason for an absence of anthocyanins remains unknown, with the flavonoid biosynthetic pathway in this flower recently being investigated. In this study, a transcriptomic approach combined with gene expression and biochemical analysis was used to predict and annotate genes in the flavonoid biosynthetic pathway. Results showed that the major flavonoid metabolites are flavonols and proanthocyanidins. Proanthocyanidin biosynthesis is exclusively catechin-based, with the gene encoding leucoanthocyanidin reductase (LAR) being well expressed. We identified 16 unigenes encoding key enzymes involved in flavonoid biosynthesis. Flavonol synthase (FLS) and dihydroflavonol reductase (DFR) appear to play important roles in regulating proanthocyanidin and flavonol levels, while anthocyanidin synthase (ANS) expression is not detected. Our results suggest that the absence of anthocyanins in Chinese narcissus is due to high expression of LAR and FLS, and a lack of ANS expression.

The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-pyrophosphate reductase, a key enzyme at the end of the methylerythritol 4-phosphate pathway (MEP), was cloned from the petals of Narcissus tazetta var. Chinensis ‘Jinzhanyintai’ and named NtHDR (accession number OQ739816). NtHDR exhibits a full-length coding sequence spanning 1380 base pairs, encoding a total of 460 amino acids. Through phylogenetic-tree analysis, it has been determined that NtHDR shared the closest evolutionary relationship with monocotyledons, specifically Asparagus officinalis and Zingiber officinale. Utilizing quantitative real-time PCR analysis, it was observed that NtHDR exhibited significant expression in both petals and corona, with expression levels varying throughout the flowering process of Narcissus. Specifically, in petals, NtHDR expression demonstrated a pattern of initial increase followed by subsequent decrease, while in corona, it consistently increased. Subsequent subcellular-localization experiments indicated that NtHDR was localized within the chloroplasts. To investigate the functional impact of NtHDR, a stable transformation was performed, where NtHDR was introduced into Nicotiana benthamiana. The resulting transgenic N. benthamiana flowers were analyzed using solid-phase microextraction coupled with gas chromatography–mass spectrometry (SPME-GC-MS) to characterize their volatile components. The analysis revealed the presence of the monoterpene compound linalool, as well as the phenylpropanoids benzyl alcohol and phenylethanol, within the floral fragrance components of the transgenic N. benthamiana plants. However, these compounds were absent in the floral fragrance components of wild-type tobacco plants, thus highlighting the impact of NtHDR transformation on the floral scent profile.

Narcissus tazetta var. chinensis M.Roem. (Chinese Narcissus) is a traditional and famous flower in China, and its corona shows a transition from green to yellow with the opening of flowers, which is of great ornamental value. To investigate the molecular mechanism of the corona color-forming in the Chinese Narcissus, in this study, a MYB gene was screened from the transcriptome of the corona by bioinformatics analysis methods, named NtMYB9. The expression level of the NtMYB9 gene was significantly higher in the corona than in the tepal, filament, ovary and leaf, and reached the highest level at the fourth period of complete coloration of the corona. The full-length sequence of the NtMYB9 coding region was cloned using the corona cDNA as a template. Sequence analysis revealed that NtMYB9 protein contained R2 and R3 domains, phylogenetic tree analysis showed that the NtMYB9 protein was most closely related to flavonol activators. Subcellular localization showed that NtMYB9 was localized in the nucleus. The overexpression of NtMYB9 gene into tobacco leaves and the expression level of seven enzyme genes related to the flavonoid biosynthesis pathway were significantly up-regulated. The transient transformation of Petunia × hybrida (Petunia) and Phalaenopsis aphrodite (Phalaenopsis) significantly revealed that the NtMYB9 gene could turn flowers yellow. These results suggest that the NtMYB9 gene is involved in the positive regulation of flavonoid biosynthesis in the Chinese Narcissus and may promote the synthesis of flavonols. In conclusion, these findings provide a valuable resource for further studies on the regulatory mechanism of the flavonoid biosynthesis pathway, and they are also beneficial to the molecular breeding of Chinese Narcissus.

Plants of the genus Narcissus are well-known for their characteristic corona morphology, which structural origins have been a bone of contention among scholars. With “Jinzhanyintai” (JZ) and “Yulinglong” (YLL)—two major close-originated cultivars of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem)—as materials, anatomic observation was made on floral organs during corona morphogenesis by dissection with hands under a stereomicroscope, paraffin section, scanning electron microscopy, and high-resolution X-ray tomography. It was uncovered that corona primordia of both cultivars appeared following the end of the differentiation of other floral organs, with differentiation sites located at the inner wall of the juncture of the base of tepals and the upper margin of the hypanthium. Affected by staminal filaments, the corona primordia of JZ experienced a three-stage differentiation process, namely blockage from the second whorl of stamens, blockage from the first whorl of stamens, and healing of corona primordia. However, the expanded spatial structure of the first whorl of petal-like stamens blocked the path of differentiation of YLL corona primordia, giving rise to slow differentiation of the corona primordia at the base of the first whorl of petal-like stamens and malformed differentiation of the corona primordia in the interval between the two whorls of petal-like stamens. Thus, a fragmented structure consisting of typical and fragmented coronas was formed. Furthermore, petal-like stamens of YLL in the lower part had a corona-like morphology. The spatio-temporal specificity of corona differentiation convincingly demonstrates that the corona is a structure independent of and different from the typical four whorls of floral organs, but also highly correlated with stamen.