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.

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.

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.

Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering.

MAIN CONCLUSION: Terpenoids play several physiological and ecological functions in plant life through direct and indirect plant defenses and also in human society because of their enormous applications in the pharmaceutical, food and cosmetics industries. Through the aid of genetic engineering its role can by magnified to broad spectrum by improving genetic ability of crop plants, enhancing the aroma quality of fruits and flowers and the production of pharmaceutical terpenoids contents in medicinal plants. Terpenoids are structurally diverse and the most abundant plant secondary metabolites, playing an important role in plant life through direct and indirect plant defenses, by attracting pollinators and through different interactions between the plants and their environment. Terpenoids are also significant because of their enormous applications in the pharmaceutical, food and cosmetics industries. Due to their broad distribution and functional versatility, efforts are being made to decode the biosynthetic pathways and comprehend the regulatory mechanisms of terpenoids. This review summarizes the recent advances in biosynthetic pathways, including the spatiotemporal, transcriptional and post-transcriptional regulatory mechanisms. Moreover, we discuss the multiple functions of the terpene synthase genes (TPS), their interaction with the surrounding environment and the use of genetic engineering for terpenoid production in model plants. Here, we also provide an overview of the significance of terpenoid metabolic engineering in crop protection, plant reproduction and plant metabolic engineering approaches for pharmaceutical terpenoids production and future scenarios in agriculture, which call for sustainable production platforms by improving different plant traits.

Transcriptome profiling provides new insights into the formation of floral scent in Hedychium coronarium.

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 ß-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.

Characterization of two monoterpene synthases involved in floral scent formation in Hedychium coronarium.

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, ß-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.

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.

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.

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.

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.

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.

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.

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'.