Search

Herein, 37 ARF genes were identified and analyzed in Hedychium coronarium and HcARF5 showed a potential role in the regulation of HcTPS3. Auxin is an important plant hormone, implicated in various aspects of plant growth and development processes especially in the biosynthesis of various secondary metabolites. Auxin response factors (ARF) belong to the transcription factors (TFs) gene family and play a crucial role in transcriptional activation/repression of auxin-responsive genes by directly binding to their promoter region. Nevertheless, whether ARF genes are involved in the regulatory mechanism of volatile compounds in flowering plants is largely unknown. β-ocimene is a key floral volatile compound synthesized by terpene synthase 3 (HcTPS3) in Hedychium coronarium. A comprehensive analysis of H. coronarium genome reveals 37 candidate ARF genes in the whole genome. Tissue-specific expression patterns of HcARFs family members were assessed using available transcriptome data. Among them, HcARF5 showed a higher expression level in flowers, and significantly correlated with the key structural β-ocimene synthesis gene (HcTPS3). Furthermore, transcript levels of both genes were associated with the flower development. Under hormone treatments, the response of HcARF5 and HcTPS3, and the emission level of β-ocimene contents were evaluated. Subcellular and transcriptional activity assay showed that HcARF5 localizes to the nucleus and possesses transcriptional activity. Yeast one-hybrid (Y1H) and dual-luciferase assays revealed that HcARF5 directly regulates the transcriptional activity of HcTPS3. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that HcARF5 interacts with scent-related HcIAA4, HcIAA6, and HcMYB1 in vivo. Overall, these results indicate that HcARF5 is potentially involved in the regulation of β-ocimene synthesis in H. coronarium.

In this study, the full cDNA sequences of HcADH2 and HcADH3 were cloned from Hedychium coronarium. The amino acid sequences encoded by them contained three most conserved motifs of short-chain alcohol dehydrogenase (ADH), namely NAD+ binding domain, TGxxx[AG]xG and active site YxxxK. The highest similarity between two genes and ADH from other plants was 70%. Phylogenetic analysis showed that they belonged to a member of the short-chain dehydrogenases/reductases 110C subfamily, but they were distinctly clustered in different clades. Real-time polymerase chain reaction analyses showed that HcADH2 was specifically expressed in bract, and it was expressed higher in no-scented Hedychium forrestii than other Hedychium species, but was undetectable in Hedychium coccineum. HcADH3 was expressed higher in the lateral petal of the flower than in other vegetative organs, and it was expressed the most in H. coronarium that is the most scented among Hedychium species, and its expression levels peaked at the half opening stage. HcADH2 and HcADH3 had almost no significant expression in leaves, but HcADH2 was expressed in response to external stimuli. The mechanical injury and methyl jasmonate (MeJA) treatment could induce expression of HcADH2 in leaves, whereas HcADH3 could have an induced expression only by MeJA. The recombinant HcADH3 protein, but not HcADH2, expressed in Escherichia coli-catalysed conversion of geraniol into citral. It was speculated that HcADH3 had an induced expression in vegetative organ of H. coronarium and took part in monoterpenoid biosynthesis in H. coronarium flowers, but the role of HcADH2 is relevant only for defensive reactions.

Floral color plays a crucial role in plant life such as plant-pollinator interactions and modifying the abiotic environment of reproductive structures. In the current study, 123 gerbera accessions were divided into six color groups (white, yellow, orange, pink, red, and purple), based on Royal Horticultural Society Color Chart calibration and colorimeter measurement. Partial least squares discriminant analysis showed that the white group was mainly affected by

The R2R3-MYB transcription factors (TFs) play several key roles in numerous plant biological processes. Hedychium coronarium is an important ornamental plant well-known for its elegant flower shape and abundant aroma type. The floral aroma of H. coronarium is due to the presence of a large amount of terpenes and benzenoids. However, less is known about the role of R2R3-MYB TFs in the regulatory mechanism of floral aroma production in this breed. Herein, we isolate and functionally characterize the R2R3-MYB TF HcMYB132, which is potentially involved in regulating floral aroma synthesis. Sequence alignment analysis revealed that it includes a nuclear localization signal NLS(s) and a 2R, 3R motif signature in the sequences. A subcellular localization assay revealed that HcMYB132 protein localizes to the nucleus. Real-time qPCR assays showed that HcMYB132 is specifically expressed in flowers and its expression pattern correlates with the emission of floral volatile compounds. In HcMYB132-silenced flowers, the levels of floral volatile compounds were significantly reduced, and the expression of key structural volatile synthesis genes was downregulated compared to control. Collectively, these results suggest that HcMYB132 might play a significant role in the regulation of terpenoid biosynthesis in H. coronarium.

Lily is one of the most economically important flowers worldwide due to its elegant appearance and appealing scent, which is mainly composed of monoterpene ocimene, linalool and benzenoids. Sugars are the primary products of plants, with fructose and hexose sugars being the substrate material for most organic compounds and metabolic pathways in plants. Herein, we isolated and functionally characterized hexokinase (LoHXK) and fructokinase (LoFRK) from Lilium 'Siberia' flower, which indicated their potential roles in floral aroma production. Real-time PCR analysis showed that LoHXK and LoFRK were highly expressed in the flower filament. Overexpression and virus-induced gene silencing (VIGS) assays revealed that LoHXK and LoFRK significantly modified the emission of β-ocimene and linalool contents via regulation of expression of key structural volatile synthesis genes (LoTPS1 and LoTPS3). Under exogenous glucose and fructose application, the volatile contents of β-ocimene and linalool were increased and the expression levels of key structural genes were upregulated. The emission of β-ocimene and linalool followed a diurnal circadian rhythm. Determination of carbon fluxes via 13C-labeled glucose and 13C-labeled fructose experiments showed that the mass spectra of ocimene and linalool significantly increased, however, the m/z ratio of ethyl benzoate did not change. Furthermore, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that LoFRK interacted with LoMYB1 and LoMYB2 proteins. Together, these results suggest that hexokinase and fructokinase may play significant roles in the regulation of ocimene and linalool biosynthesis in Lilium 'Siberia'.

An enzyme is crucial for the formation of Hedychium coronarium scent and defense responses, which may be responsible for the biosynthesis of allo-ocimene in H. coronarium. Hedychium coronarium can emit a strong scent as its main scent constituents are monoterpenes and their derivatives. Among these derivatives, allo-ocimene is not only a very important volatile substance in flower aroma, but is also crucial to plant defense. However, the molecular mechanism of allo-ocimene biosynthesis has not been characterized in plants. In this study, a new alcohol dehydrogenase gene, HcADH, was cloned. The amino acid sequences encoded by HcADH contained the most conserved motifs of short chain alcohol dehydrogenase/reductases (SDRs), which included NAD+ binding domain, TGxxx[AG]xG and active site YxxxK. Real-time PCR analyses showed that the HcADH was highly expressed in the outer labellum but was almost undetectable in vegetative organs. The change in its expression level in petals was positively correlated with the emission pattern of allo-ocimene during flower development. HcADH expression coincides also the release level of allo-ocimene among different Hedychium species. Although HcADH is not expressed in the leaves, HcADH expression and allo-ocimene release in leaves can be induced by mechanical wounding or methyl jasmonate (MeJA) treatment. In addition, the expression of HcADH induced by mechanical wounding can be prevented by acetylsalicylic acid, a jasmonic acid biosynthesis inhibitor, suggesting that jasmonic acid might participate in the transmission of wounding signals. Using the Barley stripe mosaic virus (BSMV)-VIGS method, it was found that BSMV:HcADH335 inoculation was able to down-regulate HcADH expression, decreasing only the release of allo-ocimene in flowers while the content of other volatile substances did not decrese. In vitro characterization showed that recombinant HcADH can catalyze geraniol into citral, and citral is an intermediate of allo-ocimene biosynthesis. HcADH may be responsible for the biosynthesis of allo-ocimene in H. coronarium, which is crucial for the formation of H. coronarium scent and defense function.

The SnRK (Snf1-Related protein Kinase) gene family plays crucial roles in various plant signaling pathways and stress-adaptive responses including biotic and abiotic stresses via activating protein phosphorylation pathways. However, there is no information available on the role of the SnRK gene family in Hedychium coronarium. H. coronarium is an important crop widely cultivated as an ornamental plant, herb, spice, or condiment. In this study, 60 HcSnRK genes were identified from the H. coronarium genomic and transcriptome data. Phylogenetic and gene structure analysis showed that the HcSnRK genes were divided into three groups (HcSnRK1, HcSnRK2 and HcSnRK3) and among them HcSnRK3 subfamily was further subdivided into two clades according to the number of introns. Chromosome localization analysis showed that HcSnRK genes were unevenly mapped onto all chromosomes, and the Ka/Ks ratio of 24 paralogues includes four tandems and 20 segmental duplications indicated that the HcSnRK gene family underwent a purifying selection. Cis-regulatory elements analysis suggested that the HcSnRK genes respond to multiple hormones and other stresses. The responsiveness of HcSnRK genes to several hormones was analyzed by quantitative real-time PCR. Based on the different transcriptome data, two candidates HcSnRK genes (HcSnRK2.2 and HcSnRK2.9) were screened out for further characterization . The subcellular localization experiment revealed that both genes were located in the nucleus and cytoplasm. Moreover, virus-induced gene silencing (VIGS) of HcSnRK2.2 and HcSnRK2.9 significantly reduced the floral volatile contents by suppressing the expression of terpene synthase genes (HcTPS1, HcTPS3, and HcTPS5), indicating that HcSnRK2.2 and HcSnRK2.9 genes play an important role in the regulatory mechanism of floral aroma. These results will provide novel insights into the functional dissection of H. coronarium SnRK gene family.

The MYB gene family is one of the largest groups of transcription factors (TFs) playing diverse roles in several biological processes. Hedychium coronarium (white ginger lily) is a renowned ornamental plant both in tropical and subtropical regions due to its flower shape and strong floral scent mainly composed of terpenes and benzenoids. However, there is no information available regarding the role of the MYB gene family in H. coronarium. In the current study, the MYB gene family was identified and extensively analyzed. The identified 253 HcMYB genes were unevenly mapped on 17 chromosomes at a different density. Promoter sequence analysis showed numerous phytohormones related to cis-regulatory elements. The majority of HcMYB genes contain two to three introns and motif composition analysis showed their functional conservation. Phylogenetic analysis revealed that HcMYBs could be classified into 15 distinct clades, and the segmental duplication events played an essential role in the expansion of the HcMYB gene family. Tissue-specific expression patterns of HcMYB genes displayed spatial and temporal expression. Furthermore, seven HcMYB (HcMYB7/8/75/79/145/238/248) were selected for further investigation. Through RT-qPCR, the response of candidates HcMYB genes toward jasmonic acid methyl ester (MeJA), abscisic acid (ABA), ethylene, and auxin was examined. Yeast one-hybrid (Y1H) assays revealed that candidate genes directly bind to the promoter of bottom structural volatile synthesis genes (HcTPS1, HcTPS3, HcTPS10, and HcBSMT2). Moreover, yeast two-hybrid (Y2H) assay showed that HcMYB7/8/75/145/248 interact with HcJAZ1 protein. In HcMYB7/8/79/145/248-silenced flowers, the floral volatile contents were decreased and downregulated the expression of key structural genes, suggesting that these genes might play crucial roles in floral scent formation in H. coronarium by regulating the expression of floral scent biosynthesis genes. Collectively, these findings indicate that HcMYB genes might be involved in the regulatory mechanism of terpenoids and benzenoid biosynthesis in H. coronarium.

Hedychium is an important industrial crop, which is widely cultivated for its medicinal and horticultural significance. However, the development of simple sequence repeat (SSR) for Hedychium has not yet been reported. In this study, 266,825 SSR markers were identified in H. coronarium ‘ZS’ genome. The relationship between the type, repeat times, content, and content of SSR markers and chromosome length was specifically analyzed. The results revealed that the mononucleotides (59.17 %) were the most abundant repeat motifs, followed by dinucleotides (27.97 %) and trinucleotides (10.85 %). To verify the availability of SSR and its polymorphism among different species, 120 primer pairs were randomly selected. Finally, 22 markers were screened to genotype 44 accessions collected from different geographic locations of China, Thailand, Japan, and USA. A number of 224 alleles were identified, with an average of 10.18 alleles and polymorphism information content ranged from 0.17 to 0.96 with a mean of 0.88. Genetic structure analysis indicated that 44 accessions had strong regional characteristics and a clear distinction between cultivated and wild genotypes. Population structure and PCoA analysis revealed that from 44 accessions, 7 cultivated accession were distinctly grouped into cluster A, whilst other wild accessions belong to cluster B. These new SSR markers will provide important references for genetic diversity analysis, identification of germplasm resources, and breeding of Hedychium.

Lilies are a commercially significant cut flower worldwide due not only to their elegant shape but also to their appealing scent. Among Lilium varieties, Lilium ‘Siberia’ is a cultivar that is prominent and highly favored by consumers due to its snowy white color and strong floral scent. Here, two terpene synthase genes (LoTPS2 and LoTPS4) that are responsible for floral scent production in Lilium ‘Siberia’ were cloned and functionally characterized. Recombinant LoTPS2 specifically catalyzed the formation of (E, E)-α-farnesene from FPP. Recombinant LoTPS4 is a multiproduct enzyme that produces D-limonene and β-myrcene as major volatile compounds and β-phellandrene, (+)-4-carene and 3-carene as minor products from GPP. Furthermore, LoTPS4 generates trans-α-bergamotene as a major product and di-epi-α-cedrene, α-cubebene and (E)-β-farnesene as minor compounds from FPP. Subcellular localization analysis using GFP fusion constructs revealed that LoTPS2 was localized in the cytosol, whereas LoTPS4 was localized in plastids. Real-time PCR analysis showed that LoTPS2 was highly expressed in the petals and sepals of the flower, while LoTPS4 was highly expressed in the filament of the flower. Moreover, mechanical wounding of flowers revealed that LoTPS2 showed a strong response to wounding via a rapid increase in its mRNA transcript level. Our results will assist scientists in exploring the molecular mechanisms of terpene biosynthesis in this species and will provide new insight into the biotechnological modification of the floral bouquet in Lilium.

Lilium 'Siberia' is a perennial herbaceous plant that is commercially significant because of its snowy white floral color and appealing scent which is mainly due to the presence of monoterpenes and benzoids compounds in floral volatile profile. In the current study, LoTPS5 was cloned and functionally characterized. Results revealed that LoTPS5 specifically generates squalene from FPP, whereas no product was produced when it was incubated with GPP or GGPP. The subcellular localization experiment showed that LoTPS5 was located in plastids. Furthermore, LoTPS5 showed its high expression in the leaf followed by petals and sepals of the flower. Moreover, the expression of LoTPS5 gradually increased from the bud stage and peak at the full-bloom stage. Besides, LoTPS5 showed a diurnal circadian rhythmic pattern with a peak in the afternoon (16:00) followed by deep night (24:00) and morning (8:00), respectively. LoTPS5 is highly responsive to mechanical wounding by rapidly elevating its mRNA transcript level. The current study will provide significant information for future studies of terpenoid and squalene biosynthesis in Lilium 'Siberia'.

Floral scent formation in Lilium ‘Siberia’ is mainly due to monoterpene presence in the floral profile. LoTPS1 and LoTPS3 are responsible for the formation of (±)-linalool and β-ocimene in Lilium ‘Siberia’. Lilium ‘Siberia’ is a perennial herbaceous plant belonging to Liliaceae family, cultivated both as a cut flower and garden plant. The snowy white flower emits a pleasant aroma which is mainly caused by monoterpenes present in the floral volatile profile. Previously terpene synthase (TPS) genes have been isolated and characterized from various plant species but less have been identified from Liliaceae family. Here, two terpene synthase genes (LoTPS1 and LoTPS3), which are highly expressed in sepals and petals of Lilium ‘Siberia’ flower were functionally characterized recombinant LoTPS1 specifically catalyzes the formation of (Z)-β-ocimene and (±)-linalool as its main volatile compounds from geranyl pyrophosphate (GPP), whereas LoTPS3 is a promiscuous monoterpene synthase which utilizes both GPP and farnesyl pyrophosphate (FPP) as a substrate to generate (±)-linalool and cis-nerolidol, respectively. Transcript levels of both genes were prominent in flowering parts, especially in sepals and petals which are the main source of floral scent production. The gas chromatography–mass spectrometry (GC–MS) and quantitative real-time PCR analysis revealed that the compounds were emitted throughout the day, prominently during the daytime and lower levels at night following a strong circadian rhythm in their emission pattern. Regarding mechanical wounding, both genes showed considerable involvement in floral defense by inducing the emission of (Z)-β-ocimene and (±)-linalool, elevating the transcript accumulation of LoTPS1 and LoTPS3. Furthermore, the subcellular localization experiment revealed that LoTPS1 was localized in plastids, whilst LoTPS3 in mitochondria. Our findings on these two TPSs characterized from Lilium ‘Siberia’ provide new insights into molecular mechanisms of terpene biosynthesis in this species and also provide an opportunity for biotechnological modification of floral scent profile of Lilium.

HcTPS1 and HcTPS2 are floral-scent genes that mediate terpene formation in Hedychium coronarium. Transcription of these genes is induced by various abiotic stresses and hormonal stimuli. In this study, the regulatory mechanisms responsible for HcTPS1 and HcTPS2 expression were investigated by analyzing their promoters. Two genomic fragments, 1,298-bp and 1,928-bp in length, upstream of HcTPS1 and HcTPS2, respectively, were isolated and identified as the promoters ‘PrHcTPS1’ and ‘PrHcTPS2’. Analysis of both promoter sequences indicated cis-elements associated with abiotic stress and jasmonic acid (JA) responses, as well as tissue-specific expression. To identify the functional regions within each of the two promoters, a series of 5′ deletion fragments of different lengths from each promoter were fused to a β-glucuronidase (GUS) reporter gene (pCAMBIA1300-‘prHcTPS’:: gusA) and subsequently used to transform Nicotiana tobacum L. Wisconsin (w38). Functional properties of each promoter segment were determined by GUS staining and quantitative fluorescence analyses of transgenic plants subjected to various abiotic stresses and plant hormones. GUS histochemical staining in transgenic tobacco containing the full fragment of either promoter sequence was only observed in floral organs, where gusA transcripts accumulated as flower development progressed. Additionally, gusA transcripts were also up-regulated by insect feeding, methyl jasmonate (MeJA), and wounding treatments, indicating that ‘PrHcTPS1’ and ‘PrHcTPS2’ promoters were floral-specific, developmentally-regulated, and stress-inducible. Promoter deletion analysis indicated that the promoter fragments p-420 bp of ‘PrHcTPS1’ and p-320 bp of ‘PrHcTPS2’ possessed sufficient, essential cis-elements to drive the expression patterns observed using the whole fragment of either promoter. The identified promoters could be extremely valuable for future use in molecular breeding of ornamental and horticultural plants for floral scent and stress tolerance.

Background Hedychium coronarium is a popular ornamental plant in tropical and subtropical regions because its flowers not only possess intense and inviting fragrance but also enjoy elegant shape. The fragrance results from volatile terpenes and benzenoids presented in the floral scent profile. However, in this species, even in monocots, little is known about the underlying molecular mechanism of floral scent production. Results Using Illumina platform, approximately 81 million high-quality reads were obtained from a pooled cDNA library. The de novo assembly resulted in a transcriptome with 65,591 unigenes, 50.90 % of which were annotated using public databases. Digital gene expression (DGE) profiling analysis revealed 7,796 differential expression genes (DEGs) during petal development. GO term classification and KEGG pathway analysis indicated that the levels of transcripts changed significantly in “metabolic process”, including “terpenoid biosynthetic process”. Through a systematic analysis, 35 and 33 candidate genes might be involved in the biosynthesis of floral volatile terpenes and benzenoids, respectively. Among them, flower-specific HcDXS2A, HcGPPS, HcTPSs, HcCNL and HcBCMT1 might play critical roles in regulating the formation of floral fragrance through DGE profiling coupled with floral volatile profiling analyses. In vitro characterization showed that HcTPS6 was capable of generating β-farnesene as its main product. In the transcriptome, 1,741 transcription factors (TFs) were identified and 474 TFs showed differential expression during petal development. It is supposed that two R2R3-MYBs with flower-specific and developmental expression might be involved in the scent production. Conclusions The novel transcriptome and DGE profiling provide an important resource for functional genomics studies and give us a dynamic view of biological process during petal development in H. coronarium. These data lay the basis for elucidating the molecular mechanism of floral scent formation and regulation in monocot. The results also provide the opportunities for genetic modification of floral scent profile in Hedychium. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1653-7) contains supplementary material, which is available to authorized users.

Methyl jasmonate (MeJA) is one of most abundant scent compounds in Cymbidium ensifolium flowers. In this study, the emission of MeJA and its regulation mechanism were investigated. Our results showed that emission of MeJA in C. ensifolium flowers was controlled developmentally and rhythmically. It occurred in a tissue-specific manner, and high MeJA emission was found in sepals and petals. A group of vital genes involved in the MeJA biosynthesis via the octadecanoid pathway were isolated from C. ensifolium flowers, including CeLOX, CeAOS, CeAOC and CeJMT. MeJA emission was at very low levels in unopened or half-opened C. ensifolium flowers and reached its maximal level between day 4 and 6 and declined from day 7 to 10 postanthesis. The expression of CeLOX, CeAOS, CeAOC and CeJMT increased from day 1 to day 6, and then declined from day 7 to 10 postanthesis, corresponding to the change in MeJA emission. Moreover, the expression of CeLOX, CeAOS, CeAOC and CeJMT oscillated in a rhythmic manner could reach the maximum level between 8:00 h and 16:00 h, which coincided with the MeJA emission. The high level of MeJA emission in sepals and petals coincided with the high transcript levels. The results suggest that MeJA emission in C. ensifolium flower might be directly regulated at the transcription levels. Moreover, the recombinant protein of CeJMT could specifically catalyze the jasmonic acid to form the corresponding ester MeJA.

Hedychium coronarium, a perennial herb belonging to the family Zingiberaceae, is cultivated as a garden plant or cut flower as well as for medicine and aromatic oil. Its flowers emit a fresh and inviting scent, which is mainly because of monoterpenes present in the profile of the floral volatiles. However, fragrance produced as a result of monoterpenes has not been well studied. In the present study, two novel terpene synthase (TPS) genes (HcTPS7 and HcTPS8) were isolated to study the biosynthesis of monoterpenes in H. coronarium. In vitro characterization showed that the recombinant HcTPS7 was capable of generating sabinene as its main product, in addition to nine sub-products from geranyl diphosphate (GPP). Recombinant HcTPS8 almost specifically catalyzed the formation of linalool from GPP, while it converted farnesyl diphosphate (FPP) to α-bergamotene, cis-α-bisabolene, β-farnesene and other ten sesquiterpenes. Subcellular localization experiments revealed that HcTPS7 and HcTPS8 were located in plastids. Real-time PCR analyses showed that HcTPS7 and HcTPS8 genes were highly expressed in petals and sepals, but were almost undetectable in vegetative organs. The changes of their expression levels in petals were positively correlated with the emission patterns of sabinene and linalool, respectively, during flower development. The results indicated that HcTPS7 and HcTPS8 were involved in the biosynthesis of sabinene and linalool in H. coronarium flowers. Results on these two TPSs first characterized from H. coronarium provide new insights into molecular mechanisms of terpene biosynthesis in this species and also lay the basis for biotechnological modification of floral scent profile in Hedychium.

Farnesyl pyrophosphate synthase (FPPS EC 2.5.1.10) catalyzes the production of farnesyl pyrophosphate (FPP), which is a key precursor for many sesquiterpenoids such as floral scent and defense volatiles against herbivore attack. Here we report a new full-length cDNA encoding farnesyl diphosphate synthase from Hedychium coronarium. The open reading frame for full-length HcFPPS encodes a protein of 356 amino acids, which is 1068 nucleotides long with calculated molecular mass of 40.7 kDa. Phylogenetic tree analysis indicates that HcFPPS belongs to the plant FPPS super-family and has strong relationship with FPPS from Musa acuminata. Expression of the HcFPPS gene in Escherichia coli yielded FPPS activity. Tissue-specific and developmental analyses of the HcFPPS mRNA and corresponding volatile sesquiterpenoid levels in H. coronarium flowers revealed that the HcFPPS might play a regulatory role in floral volatile sesquiterpenoid biosynthesis. The emission of the FPP-derived volatile terpenoid correlates with strong expression of HcFPPS induced by mechanical wounding and Udaspes folus-damage in leaves, which suggests that HcFPPS may have an important ecological function in H. coronarium vegetative organ.