Comparative metabolomics analysis reveals dynamic changes in carbohydrate profiles of corms during the "relay growth" of konjac (Amorphophallus muelleri).

The type and content of carbohydrates in konjac corms are an essential factors in determining the quality of konjac; however, the pattern of carbohydrate changes and the mechanism regulating the development of mother and daughter corms in the "relay growth" process of Amorphophallus muelleri remain unclear. This study aimed to investigate changes in corm carbohydrates during the growth cycle of A. muelleri and to compare the carbohydrate composition and the expression of related genes between mother and daughter corms. Integrated metabolome and RNA-seq analyses identified 37 differential metabolites as well as 8074 genes that were differentially expressed between mother and daughter corms, the majority of which were involved in starch and sucrose metabolism. More than 80% of the differential metabolites, including sucrose and starch, tended to accumulate in the mother corms; however, konjac glucomannan (KGM), as one of the most important carbohydrates and its major component of the corm, accumulated in higher amounts in the daughter corms. In addition, the expression of invertase and alpha-amylase that promote the breakdown of sucrose and starch was 351.78- and 15.63-fold higher, respectively, in the daughter corm, whereas that of the starch synthesis gene AkWAXY was only 0.096 times as high as in the mother corms. Furthermore, the level of cellulose synthase-like protein G, which promotes KGM synthesis, was 3.85 times higher in daughter corms compared to mother corms. Thus, we inferred that the daughter and mother corms had two distinct carbohydrate utilization strategies. This study provides insights into temporal changes in carbohydrates during the growth cycle of A. muelleri.

Aroma Components in Horticultural Crops: Chemical Diversity and Usage of Metabolic Engineering for Industrial Applications.

Plants produce an incredible variety of volatile organic compounds (VOCs) that assist the interactions with their environment, such as attracting pollinating insects and seed dispersers and defense against herbivores, pathogens, and parasites. Furthermore, VOCs have a significant economic impact on crop quality, as well as the beverage, food, perfume, cosmetics and pharmaceuticals industries. These VOCs are mainly classified as terpenoids, benzenoids/phenylpropanes, and fatty acid derivates. Fruits and vegetables are rich in minerals, vitamins, antioxidants, and dietary fiber, while aroma compounds play a major role in flavor and quality management of these horticultural commodities. Subtle shifts in aroma compounds can dramatically alter the flavor and texture of fruits and vegetables, altering their consumer appeal. Rapid innovations in -omics techniques have led to the isolation of genes encoding enzymes involved in the biosynthesis of several volatiles, which has aided to our comprehension of the regulatory molecular pathways involved in VOC production. The present review focuses on the significance of aroma volatiles to the flavor and aroma profile of horticultural crops and addresses the industrial applications of plant-derived volatile terpenoids, particularly in food and beverages, pharmaceuticals, cosmetics, and biofuel industries. Additionally, the methodological constraints and complexities that limit the transition from gene selection to host organisms and from laboratories to practical implementation are discussed, along with metabolic engineering's potential for enhancing terpenoids volatile production at the industrial level.

Integrated metabolome and transcriptome analysis provides insights on the floral scent formation in Hydrangea arborescens.

Hydrangea (Hydrangea arborescens var. "Annabelle") flowers are composed of sweet aroma sepals rather than true petals and can change color. Floral volatiles plays important roles in plants, such as attracting pollinators, defending against herbivores, and signaling. However, the biosynthesis and regulatory mechanisms underlying fragrance formation in H. arborescens during flower development remain unknown. In this study, a combination of metabolite profiling and RNA sequencing (RNA-seq) was employed to identify genes associated with floral scent biosynthesis mechanisms in "Annabelle" flowers at three developmental stages (F1, F2, and F3). The floral volatile data revealed that the "Annabelle" volatile profile includes a total of 33 volatile organic compounds (VOCs), and VOCs were abundant during the F2 stage of flower development, followed by the F1 and F3 stages, respectively. Terpenoids and benzenoids/phenylpropanoids were abundant during the F2 and F1 stages, with the latter being the most abundant, whereas fatty acid derivatives and other compounds were found in large amounts during the F3 stage. According to ultra-performance liquid chromatography-tandem mass spectrometer analysis, benzene and substituted derivatives, carboxylic acids and derivatives, and fatty acyls play a significant role in the floral metabolite profile. The transcriptome data revealed a total of 17,461 differentially expressed genes (DEGs), with 7585, 12,795, and 9044 DEGs discovered between the F2 and F1, F3 and F1, and F2 and F3 stages, respectively. Several terpenoids and benzenoids/phenylpropanoids biosynthesis-related DEGs were identified, and GRAS/bHLH/MYB/AP2/WRKY were more abundant among transcription factors. Finally, DEGs interlinked with VOCs compounds were determined using Cytoscape and k-means analysis. Our results pave the way for the discovery of new genes, critical data for future genetic studies, and a platform for the metabolic engineering of genes involved in the production of Hydrangea's signature floral fragrance.

Volatile organic compounds as mediators of plant communication and adaptation to climate change.

Plant volatile organic compounds are the most abundant and structurally diverse plant secondary metabolites. They play a key role in plant lifespan via direct and indirect plant defenses, attracting pollinators, and mediating various interactions between plants and their environment. The ecological diversity and context-dependence of plant-plant communication driven by volatiles are crucial elements that influence plant performance in different habitats. Plant volatiles are also valued for their multiple applications in food, flavor, pharmaceutical, and cosmetics industries. In the current review, we summarize recent advances that have elucidated the functions of plant volatile organic compounds as mediators of plant interaction at community and individual levels, highlighting the complexities of plant receiver feedback to various signals and cues. This review emphasizes volatile terpenoids, the most abundant class of plant volatile organic compounds, highlighting their role in plant adaptability to global climate change and stress-response pathways that are integral to plant growth and survival. Finally, we identify research gaps and suggest future research directions.

A high trans-zeatin nucleoside concentration in corms may promote the multileaf growth of Amorphophallus muelleri.

Amorphophallus muelleri has a multileaf growth pattern different from that of other konjacs; however, the hormonal mechanism underlying this phenomenon is not clear. In this study, the levels of hormones closely related to the sprouting of the axillary bud, including five types of cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) were measured. In the second leaf sprouting stage, the content of trans-zeatin riboside (tZR) in corms increased more than 5000-fold over that in the dormancy period. Surprisingly, although the expression of CYP735A1 and CYP735A2, which synthesize the precursors for tZR was elevated at the second leaf sprouting stage, the expression of IPTs, which have key roles in cytokinin biosynthesis, did not change significantly. In addition, most cytokinin contents in leaves during the same period were significantly lower than those in corms. We speculate that the high cytokinin contents in the corms may not biosynthesized de novo in corms. In addition, the IAA content in the corms also considerably increased during the second leaf sprouting stage. Indole-3-acetaldehyde oxidase (AO1) and auxin efflux carrier PIN1A, presented relatively high expression levels in the same period. In contrast, ABA content, and the expression of NCED1, a rate-limiting enzyme in ABA biosynthesis, were suppressed at the second leaf sprouting stage. It is worth mentioning that N6-(Δ2-isopentenyl) adenosine (iP)-type cytokinins have a high content in corms in the dormant period that significantly decreases after the first leaf sprouting stage, which is completely different from the trend of tZR. By treating dormant corms with iP, the percentage of multibud plants increased, and the growth performance in terms of bud and root length was significantly higher than those of the control. This implies that iP-type cytokinins tend to play a role in promoting first seedling sprouting. Furthermore, there was a remarkable increase of the IAA content in both corms and roots under iP treatment but an inhibitory effect in buds. We speculate that the increase in the IAA content induced by iP is tissue specific. These results will assist in the understanding of the role of hormones, especially cytokinins, in the multileaf growth type of konjac.

Molecular cloning, characterization and expression analysis of two 12-oxophytodienoate reductases (NtOPR1 and NtOPR2) from Nicotiana tabacum.

BACKGROUND: 12-oxophytodienoic acid (OPDA) is a signaling molecule involved in defense and stress responses in plants. 12-oxophytodienoate reductase (OPR) is involved in the biosynthesis of jasmonic acid and trigger the conversion of OPDA into 3-oxo-2(2'[Z]-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0). METHODS AND RESULTS: Sequence analysis revealed that Nicotiana tabacum 12-oxophytodienoate reductase 1 (OPR1) and OPR2 encoded polypeptides of 375 and 349 amino acids with molecular masses of 41.67 and 39.04 kilodaltons (kDa), respectively, while the deduced protein sequences of NtOPR1 and NtOPR2 showed high homology with other 12-oxophytodienoate reductases. BLAST (Basic local alignment search tool) analysis revealed that both NtOPRs belong to the family of Old Yellow Enzymes (OYE), and analysis of genomic DNA structure indicated that both genes include 5 exons and 4 introns. Phylogenetic analysis using MEGA X showed that NtOPR1 and NtOPR2 shared a close evolutionary relationship with Nicotiana attenuata 12-oxophytodienoate reductases. In silico analysis of subcellular localization indicated the probable locations of NtOPR1 and NtOPR2 to be the cytoplasm and the peroxisome, respectively. Tissue-specific expression assays via qRT-PCR revealed that NtOPR1 and NtOPR2 genes were highly expressed in Nicotiana tabacum roots, temperately expressed in leaves and flowers, while low expression was observed in stem tissue. CONCLUSIONS: Presently, two 12-oxophytodienoate reductase genes (NtOPR1 and NtOPR2) were cloned and comprehensively characterized. Our findings provide comprehensive analyses that may guide future deep molecular studies of 12-oxophytodienoate reductases in Nicotiana tabacum.

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'.

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.

Identification, methylation profiling, and expression analysis of stress-responsive cytochrome P450 genes in rice under abiotic and phytohormones stresses.

The cytochrome P450 (CYP) is a large and complex eukaryotic gene superfamily with enzymatic activities involved in several physiological and regulatory processes. As an objective, an in-silico genome-wide DNA methylation (5mC) analysis was performed in rice (Oryza sativa cv. Zhonghua11), and the epigenetic role of CYPs in two abiotic stresses was observed. Being a stable representative mark, DNA-methylation alters the gene expression under stressful environmental conditions. Rice plants under salinity and drought stresses were analyzed through MeDIP-chip hybridization, and 14 unique genes of the CYP family were identified in the rice genome with varying degrees of methylation. The gene structure, promoter sequences, and phylogenetic analysis were performed. Furthermore, the responses of CYPs to various abiotic stresses, including salinity, drought, and cold were revealed. Similarly, the expression profile of potential CYPs was also investigated under various phytohormone stresses, which revealed the potential involvement of CYPs to hormone regulations. Overall, the current study provides evidence for CYP's stress regulation and fundamental for further characterization and understanding their epigenetic roles in gene expression regulation and environmental stress regulation in higher plants.

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.

The prohibitins (PHB) gene family in tomato: Bioinformatic identification and expression analysis under abiotic and phytohormone stresses.

The prohibitins (PHB) are SPFH domain-containing proteins found in the prokaryotes to eukaryotes. The plant PHBs are associated with a wide range of biological processes, including senescence, development, and responses to biotic and abiotic stresses. The PHB proteins are identified and characterized in the number of plant species, such as Arabidopsis, rice, maize, and soybean. However, no systematic identification of PHB proteins was performed in Solanum lycopersicum. In this study, we identified 16 PHB proteins in the tomato genome. The analysis of conserved motifs and gene structure validated the phylogenetic classification of tomato PHB proteins. It was observed that various members of tomato PHB proteins undergo purifying selection based on the Ka/Ks ratio and are targeted by four families of miRNAs. Moreover, SlPHB proteins displayed a very unique expression pattern in different plant parts including fruits at various development stages. It was found that SlPHBs processed various development-related and phytohormone responsive cis-regulatory elements in their promoter regions. Furthermore, the exogenous phytohormones treatments (Abscisic acid, indole-3-acetic acid, gibberellic acid, methyl jasmonate) salt and drought stresses induce the expression of SlPHB. Moreover, the subcellular localization assay revealed that SlPHB5 and SlPHB10 were located in the mitochondria. This study systematically summarized the general characterization of SlPHBs in the tomato genome and provides a foundation for the functional characterization of PHB genes in tomato and other plant species.

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.

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.

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.

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.