Identification and functional analysis of floral terpene synthase genes in Curcuma alismatifolia.

MAIN CONCLUSION: CaTPS2 and CaTPS3 were significantly expressed in flowers of Curcuma alismatifolia 'Shadow' and demonstrated bifunctional enzyme activity, CaTPS2 generated linalool and nerolidol as products, and CaTPS3 catalyzed ß-myrcene and ß-farnesene formation. This study presents the discovery and functional characterization of floral terpene synthase (TPS) genes in Curcuma alismatifolia 'Shadow', a cultivar renowned for its unique fragrance. Addressing the gap in understanding the genetic basis of floral scent in this species, we identified eight TPS genes through comprehensive transcriptome sequencing. Among these, CaTPS2 and CaTPS3 were significantly expressed in floral tissues and demonstrated bifunctional enzyme activity corresponding to the major volatile compounds detected in 'Shadow'. Functional analyses, including in vitro assays complemented with rigorous controls and alternative identification methods, elucidated the roles of these TPS genes in terpenoid biosynthesis. In vitro studies were conducted via heterologous expression in E. coli, followed by purification of the recombinant protein using affinity chromatography, enzyme assays were performed with GPP/FPP as the substrate, and volatile products were inserted into the GC-MS for analysis. Partially purified recombinant protein of CaTPS2 catalyzed GPP and FPP to produce linalool and nerolidol, respectively, while partially purified recombinant protein of CaTPS3 generated ß-myrcene and ß-farnesene with GPP and FPP as substrates, respectively. Real-time quantitative PCR further validated the expression patterns of these genes, correlating with terpenoid accumulation in different plant tissues. Our findings illuminate the molecular mechanisms underpinning floral fragrance in C. alismatifolia and provide a foundation for future genetic enhancements of floral scent in ornamental plants. This study, therefore, contributes to the broader understanding of terpenoid biosynthesis in plant fragrances, paving the way for biotechnological applications in horticulture plant breeding.

A BAHD acyltransferase contributes to the biosynthesis of both ethyl benzoate and methyl benzoate in the flowers of Lilium oriental hybrid 'Siberia'.

Lily is a popular flower worldwide due to its elegant appearance and pleasant fragrance. Floral volatiles of lily are predominated by monoterpenes and benzenoids. While a number of genes for monoterpene biosynthesis have been characterized, the molecular mechanism underlying floral benzenoid formation in lily remains unclear. Here, we report on the identification and characterization of a novel BAHD acyltransferase gene that contributes to the biosynthesis of two related floral scent benzoate esters, ethyl benzoate and methyl benzoate, in the scented Lilium oriental hybrid 'Siberia'. The emission of both methyl benzoate and ethyl benzoate in L. 'Siberia' was found to be tepal-specific, floral development-regulated and rhythmic. Through transcriptome profiling and bioinformatic analysis, a BAHD acyltransferase gene designated LoAAT1 was identified as the top candidate gene for the production of ethyl benzoate. In vitro enzyme assays and substrate feeding assays provide substantial evidence that LoAAT1 is responsible for the biosynthesis of ethyl benzoate. It was interesting to note that in in vitro enzyme assay, LoAAT1 can also catalyze the formation of methyl benzoate, which is typically formed by the action of benzoic acid methyltransferase (BAMT). The lack of an expressed putative BAMT gene in the flower transcriptome of L. 'Siberia', together with biochemical and expression evidence, led us to conclude that LoAAT1 is also responsible for, or at least contributes to, the biosynthesis of the floral scent compound methyl benzoate. This is the first report that a member of the plant BAHD acyltransferase family contributes to the production of both ethyl benzoate and methyl benzoate, presenting a new mechanism for the biosynthesis of benzoate esters.

Light Regulation of LoCOP1 and Its Role in Floral Scent Biosynthesis in Lilium 'Siberia'.

Light is an important environmental signal that governs plant growth, development, and metabolism. Constitutive photomorphogenic 1 (COP1) is a light signaling component that plays a vital role in plant light responses. We isolated the COP1 gene (LoCOP1) from the petals of Lilium 'Siberia' and investigated its function. The LoCOP1 protein was found to be the most similar to Apostasia shenzhenica COP1. LoCOP1 was found to be an important factor located in the nucleus and played a negative regulatory role in floral scent production and emission using the virus-induced gene silencing (VIGS) approach. The yeast two-hybrid, ß-galactosidase, and bimolecular fluorescence complementation (BiFC) assays revealed that LoCOP1 interacts with LoMYB1 and LoMYB3. Furthermore, light modified both the subcellular distribution of LoCOP1 and its interactions with LoMYB1 and MYB3 in onion cells. The findings highlighted an important regulatory mechanism in the light signaling system that governs scent emission in Lilium 'Siberia' by the ubiquitination and degradation of transcription factors via the proteasome pathway.

Identification and Characterization of Jasmonic Acid Methyltransferase Involved in the Formation of Floral Methyl Jasmonate in Hedychium coronarium.

Hedychium coronarium is a popular ornamental flower in tropical and subtropical areas due to its elegant appearance and inviting fragrance. Methyl jasmonate (MeJA) is one of the volatile compounds in the blooming flowers of H. coronarium. However, the molecular mechanism underlying floral MeJA formation is still unclear in H. coronarium. In this study, a total of 12 SABATH family genes were identified in the genome of H. coronarium, and their encoded proteins range from 366 to 387 amino acids. Phylogenetic analysis revealed seven clades in the SABATH family and a JMT ortholog clade, including two HcSABATH members. Combined with expression profiling of HcSABATH members, HcJMT1 was identified as the top candidate gene for floral MeJA biosynthesis. In vitro enzyme assays showed that HcJMT1 can catalyze the production of MeJA from jasmonic acid. Gene expression analysis indicated that HcJMT1 exhibited the highest expression in the labella and lateral petals, the major sites of MeJA emission. During flower development, the two MeJA isomers, major isomers in the products of the HcJMT1 protein, were released after anthesis, in which stage HcJMT1 displayed high expression. Our results indicated that HcJMT1 is involved in the formation of floral MeJA in H. coronarium.

Phenotypic Variation in Flower Color and Morphology in the Gerbera (Gerbera hybrida) F1 Hybrid Population and Their Association with EST-SSR Markers.

Gerbera (Gerbera hybrida) is a widely cultivated ornamental plant. However, its genetic improvement is limited by the lack of genetic analysis and molecular markers for traits. In this study, we analyzed the phenotypic and genotypic variation of 140 F1 progeny from two gerbera varieties with different flower types and colors. We evaluated the flower's morphology, color, and pigment content of the F1 population and performed cluster principal component analysis (PCA) and correlation analysis. The results showed that the main ornamental traits of the hybrid progeny varied greatly. The segregation ratios of single and double flowers and ligulate and split ray florets were both 1:1. The flower colors of the F1 progeny were mainly red and purple-red, similar to the male parent's color. Furthermore, we conducted a genetic analysis of the hybrid progeny using EST-SSR markers and performed association analysis with phenotypic traits. We identified 2, 2, 3, 1, and 2 loci to be associated with peduncle length (PL), ray floret length (RFL), and outer ray floret; the level of apex relative to the top of involucre (LAI); outer corolla lips (OCL); and the b* of ray floret color, respectively. Our results reveal the genetic patterns of important ornamental traits and provide a theoretical basis and practical tools for gerbera genetic breeding.

Functional Characterization of Hedychium coronarium J. Koenig MYB132 Confers the Potential Role in Floral Aroma Synthesis.

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.

Putative regulatory role of hexokinase and fructokinase in terpenoid aroma biosynthesis in Lilium 'Siberia'.

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 ß-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 ß-ocimene and linalool were increased and the expression levels of key structural genes were upregulated. The emission of ß-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'.

HS-SPME-GC-MS and Electronic Nose Reveal Differences in the Volatile Profiles of Hedychium Flowers.

Floral fragrance is one of the most important characteristics of ornamental plants and plays a pivotal role in plant lifespan such as pollinator attraction, pest repelling, and protection against abiotic and biotic stresses. However, the precise determination of floral fragrance is limited. In the present study, the floral volatile compounds of six Hedychium accessions exhibiting from faint to highly fragrant were comparatively analyzed via gas chromatography-mass spectrometry (GC-MS) and Electronic nose (E-nose). A total of 42 volatile compounds were identified through GC-MS analysis, including monoterpenoids (18 compounds), sesquiterpenoids (12), benzenoids/phenylpropanoids (8), fatty acid derivatives (2), and others (2). In Hedychium coronarium 'ZS', H. forrestii 'Gaoling', H. 'Jin', H. 'Caixia', and H. 'Zhaoxia', monoterpenoids were abundant, while sesquiterpenoids were found in large quantities in H. coccineum 'KMH'. Hierarchical clustering analysis (HCA) divided the 42 volatile compounds into four different groups (I, II, III, IV), and Spearman correlation analysis showed these compounds to have different degrees of correlation. The E-nose was able to group the different accessions in the principal component analysis (PCA) corresponding to scent intensity. Furthermore, the pattern-recognition findings confirmed that the E-nose data validated the GC-MS results. The partial least squares (PLS) analysis between floral volatile compounds and sensors suggested that specific sensors were highly sensitive to terpenoids. In short, the E-nose is proficient in discriminating Hedychium accessions of different volatile profiles in both quantitative and qualitative aspects, offering an accurate and rapid reference technique for future applications.

Auxin-Responsive R2R3-MYB Transcription Factors HcMYB1 and HcMYB2 Activate Volatile Biosynthesis in Hedychium coronarium Flowers.

Auxin, an important plant hormone, induces the biosynthesis of various secondary metabolites by modulating the expression of auxin-responsive genes. In the ornamental plant Hedychium coronarium, linalool and methyl benzoate are biosynthesized by the terpene synthase (TPS) HcTPS5 and the benzoic/salicylic acid methyltransferase (BSMT) HcBSMT2, respectively. However, the transcriptional regulation of this process remains unclear. Here, we identified and functionally characterized the R2R3-MYB transcription factors HcMYB1 and HcMYB2 in regulating the biosynthesis of these floral aroma compounds. HcMYB1 and HcMYB2 are specifically expressed in flowers, their expression is correlated with the emission of volatile compounds in flowers, and is induced by auxin. Moreover, HcMYB1 and HcMYB2 interact with the HcBSMT2 promoter region. HcMYB2 activates the expression of the linalool synthase gene HcTPS5. In flowers with HcMYB1 or HcMYB2 silenced, the levels of floral scent compounds were significantly reduced, and HcBSMT2 and HcTPS5 were downregulated compared with the wild type. Moreover, HcMYB1 form protein-protein interaction with key scent-related HcIAA4 protein to regulate floral aroma production. Taken together, these results indicate that HcMYB1 and HcMYB2 play crucial roles in regulating the formation of scent compounds in Hedychium coronarium (H. coronarium) flowers in response to auxin signaling.

Genome-wide analysis of ARF transcription factors reveals HcARF5 expression profile associated with the biosynthesis of ß-ocimene synthase in Hedychium coronarium.

KEY MESSAGE: 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. ß-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 ß-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 ß-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 ß-ocimene synthesis in H. coronarium.

Genome-wide identification and expression pattern of SnRK gene family under several hormone treatments and its role in floral scent emission in Hedychium coronarium.

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.

Coordinated and High-Level Expression of Biosynthetic Pathway Genes Is Responsible for the Production of a Major Floral Scent Compound Methyl Benzoate in Hedychium coronarium.

Methyl benzoate is a constituent of floral scent profile of many flowering plants. However, its biosynthesis, particularly in monocots, is scarcely reported. The monocot Hedychium coronarium is a popular ornamental plant in tropical and subtropical regions partly for its intense and inviting fragrance, which is mainly determined by methyl benzoate and monoterpenes. Interestingly, several related Hedychium species lack floral scent. Here, we studied the molecular mechanism of methyl benzoate biosynthesis in H. coronarium. The emission of methyl benzoate in H. coronarium was found to be flower-specific and developmentally regulated. As such, seven candidate genes associated with methyl benzoate biosynthesis were identified from flower transcriptome of H. coronarium and isolated. Among them, HcBSMT1 and HcBSMT2 were demonstrated to catalyze the methylation of benzoic acid and salicylic acid to form methyl benzoate and methyl salicylate, respectively. Methyl salicylate is a minor constituent of H. coronarium floral scent. Kinetic analysis revealed that HcBSMT2 exhibits a 16.6-fold lower Km value for benzoic acid than HcBSMT1, indicating its dominant role for floral methyl benzoate formation. The seven genes associated with methyl benzoate biosynthesis exhibited flower-specific or flower-preferential expression that was developmentally regulated. The gene expression and correlation analysis suggests that HcCNL and HcBSMT2 play critical roles in the regulation of methyl benzoate biosynthesis. Comparison of emission and gene expression among four Hedychium species suggested that coordinated and high-level expression of biosynthetic pathway genes is responsible for the massive emission of floral methyl benzoate in H. coronarium. Our results provide new insights into the molecular mechanism for methyl benzoate biosynthesis in monocots and identify useful molecular targets for genetic modification of scent-related traits in Hedychium.

Genome-Wide Analysis Reveals the Potential Role of MYB Transcription Factors in Floral Scent Formation in Hedychium coronarium.

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.

Metabolite and Transcriptome Profiling Analysis Revealed That Melatonin Positively Regulates Floral Scent Production in Hedychium coronarium.

Melatonin is a pleiotropic molecule that regulates a variety of developmental processes. Floral volatiles are important features of flowers that facilitate flower-visitor interactions by attracting pollinators, structure flower-visitor communities, and play defensive roles against plant and flower antagonists. Aside from their role in plants, floral volatiles are an essential ingredient in cosmetics, perfumes, pharmaceuticals, and flavorings. Herein, integrated metabolomic and transcriptomic approaches were carried out to analyze the changes triggered by melatonin exposure during the Hedychium coronarium flower development stages. Quantitative analysis of the volatiles of H. coronarium flowers revealed that volatile organic compound emission was significantly enhanced after melatonin exposure during the half bloom (HS), full bloom (FB) and fade stage (FS). Under the melatonin treatment, the emission of volatile contents was highest during the full bloom stage of the flower. Variable importance in projection (VIP) analysis and partial least-squares discriminant analysis (PLS-DA) identified 15 volatile compounds with VIP > 1 that were prominently altered by the melatonin treatments. According to the transcriptome sequencing data of the HS, FB, and FS of the flowers, 1,372, 1,510, and 1,488 differentially expressed genes were identified between CK-HS and 100MT-HS, CK-FB and 100MT-FB, and CK-FS and 100MT-FS, respectively. Among the significant differentially expressed genes (DEGs), 76 were significantly upregulated and directly involved in the floral scent biosynthesis process. In addition, certain volatile organic compounds were substantially linked with various DEGs after combining the metabolome and transcriptome datasets. Moreover, some transcription factors, such as MYB and bHLH, were also significantly upregulated in the comparison, which might be related to the floral aroma mechanism. Our results suggested that melatonin increased floral aroma production in H. coronarium flowers by modifying the expression level of genes involved in the floral scent biosynthesis pathway. These findings serve as a foundation for future research into the molecular mechanisms underlying the dynamic changes in volatile contents induced by melatonin treatment in H. coronarium.

Classification and Association Analysis of Gerbera (Gerbera hybrida) Flower Color Traits.

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 L* value, a* value, C value, and total anthocyanin contents, while the yellow group was positively correlated with L* value, b* value, and total anthocyanin contents. Similarly, the orange group was mainly affected by b* value and total carotenoid contents, whereas the pink group was positively correlated with L* and h values. Furthermore, the red group was affected by L* value, a* value, C value, and total anthocyanin contents, whilst the purple group was mainly distributed by L* value, a* value, b* value, and total anthocyanin contents. Based on 'Jin Xiang' transcriptome data, 14,106 expressed sequence tag (EST)-SSR markers were identified and 48 pairs of primers (19 newly developed primers) were screened. Population genetic structure, neighbor-joining clustering, and principal coordinate analysis showed that 123 gerbera accessions could be divided into two groups. EST-SSR-based association analysis showed that 1, 1, 2, 1, 1, 2, and 1 significant loci were related to L*, a*, b*, C, and h, total carotenoid, and total anthocyanin contents, respectively. These results provide an important reference for flower color classification and genetic improvement of gerbera.

Cloning, functional characterization and expression analysis of LoTPS5 from Lilium 'Siberia'.

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'.

Molecular cloning, characterization and expression analysis of LoTPS2 and LoTPS4 involved in floral scent formation in oriental hybrid Lilium variety 'Siberia'.

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 ß-myrcene as major volatile compounds and ß-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)-ß-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.

A Hedychium coronarium short chain alcohol dehydrogenase is a player in allo-ocimene biosynthesis.

KEY MESSAGE: 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.

Genome-Wide Analysis and Characterization of the Aux/IAA Family Genes Related to Floral Scent Formation in Hedychium coronarium.

Auxin plays a key role in different plant growth and development processes, including flower opening and development. The perception and signaling of auxin depend on the cooperative action of various components, among which auxin/indole-3-acetic acid (Aux/IAA) proteins play an imperative role. In a recent study, the entire Aux/IAA gene family was identified and comprehensively analyzed in Hedychium coronarium, a scented species used as an ornamental plant for cut flowers. Phylogenetic analysis showed that the Aux/IAA gene family in H. coronarium is slightly contracted compared to Arabidopsis, with low levels of non-canonical proteins. Sequence analysis of promoters showed numerous cis-regulatory elements related to various phytohormones. HcIAA genes showed distinct expression patterns in different tissues and flower developmental stages, and some HcIAA genes showed significant responses to auxin and ethylene, indicating that Aux/IAAs may play an important role in linking hormone signaling pathways. Based on the expression profiles, HcIAA2, HcIAA4, HcIAA6 and HcIAA12, were selected as candidate genes and HcIAA2 and HcIAA4 were screened for further characterization. Downregulation of HcIAA2 and HcIAA4 by virus-induced gene silencing in H. coronarium flowers modified the total volatile compound content, suggesting that HcIAA2 and HcIAA4 play important roles in H. coronarium floral scent formation. The results presented here will provide insights into the putative roles of HcIAA genes and will assist the elucidation of their precise roles during floral scent formation.

Functional characterization and expression analysis of two terpene synthases involved in floral scent formation in Lilium 'Siberia'.

MAIN CONCLUSION: 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 ß-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)-ß-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)-ß-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.