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.

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.

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.

Ramet specialization allows similar performance of the invasive macrophyte Hedychium coronarium under different light and intraspecific competition levels

Invasive species can affect ecosystems functioning by forming dense monospecific stands and outcompeting native plants. However, the performance of the invader depends on its plastic responses to abiotic attributes of invaded communities. Understanding the interplay between intraspecific competition and environmental conditions is important to elucidate the domain and aggressive potential of invasive species. Here, we assessed the performance of the invasive Hedychium coronarium in two levels of intraspecific competition created through rhizome density under full light and partial shade. We tested the influence of light, density and their interaction on ramet length and number of ramets during the first three months after planting (phase 1) and after 22 months (phase 2), and on rhizome dry weight and the ramet/rhizome biomass ratio in phase 2. In both phases, ramets were longer under shade and the number of ramets was higher under low rhizome density indicating lower intraspecific competition. In phase 2, there was a negative effect of rhizome density on ramet length, but it was restricted to shade, probably due to the aggravation of competition for light. Rhizome dry weight was greater under shade conditions and it was not affected by rhizome density, and ramet/rhizome ratio did not differ between treatments. Our results supported a large phenotypic plasticity of H. coronarium ramets, which allowed similar performances despite variations in environmental conditions and population densities. We suggest that this mechanism of energy allocation enhances the success of this invasive plant in varied habitats, such as open and closed, forested sites.

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.