Volatile Organic Compounds from Orchids: From Synthesis and Function to Gene Regulation.

Orchids are one of the most significant plants that have ecologically adapted to every habitat on earth. Orchids show a high level of variation in their floral morphologies, which makes them popular as ornamental plants in the global market. Floral scent and color are key traits for many floricultural crops. Volatile organic compounds (VOCs) play vital roles in pollinator attraction, defense, and interaction with the environment. Recent progress in omics technology has led to the isolation of genes encoding candidate enzymes responsible for the biosynthesis and regulatory circuits of plant VOCs. Uncovering the biosynthetic pathways and regulatory mechanisms underlying the production of floral scents is necessary not only for a better understanding of the function of relevant genes but also for the generation of new cultivars with desirable traits through molecular breeding approaches. However, little is known about the pathways responsible for floral scents in orchids because of their long life cycle as well as the complex and large genome; only partial terpenoid pathways have been reported in orchids. Here, we review the biosynthesis and regulation of floral volatile compounds in orchids. In particular, we focused on the genes responsible for volatile compounds in various tissues and developmental stages in Cymbidium orchids. We also described the emission of orchid floral volatiles and their function in pollination ecology. Taken together, this review will provide a broad scope for the study of orchid floral scents.

RNA sequencing analysis of Cymbidium goeringii identifies floral scent biosynthesis related genes.

BACKGROUND: Cymbidium goeringii belongs to the Orchidaceae, which is one of the most abundant angiosperm families. Cymbidium goeringii consist with high economic value and characteristics include fragrance and multiple flower colors. Floral scent is one of the important strategies for ensuring fertilization. However, limited genetic data is available in this non-model plant, and little known about the molecular mechanism responsible for floral scent in this orchid. Transcriptome and expression profiling data are needed to identify genes and better understand the biological mechanisms of floral scents in this species. Present transcriptomic data provides basic information on the genes and enzymes related to and pathways involved in flower secondary metabolism in this plant. RESULTS: In this study, RNA sequencing analyses were performed to identify changes in gene expression and biological pathways related scent metabolism. Three cDNA libraries were obtained from three developmental floral stages: closed bud, half flowering stage and full flowering stage. Using Illumina technique 159,616,374 clean reads were obtained and were assembled into 85,868 final unigenes (average length 1194 nt), 33.85% of which were annotated in the NCBI non redundant protein database. Among this unigenes 36,082 were assigned to gene ontology and 23,164 were combined with COG groups. Total 33,417 unigenes were assigned in 127 pathways according to the Kyoto Encyclopedia of Genes and Genomes pathway database. According these transcriptomic data we identified number of candidates genes which differentially expressed in different developmental stages of flower related to fragrance biosynthesis. In q-RT-PCR most of the fragrance related genes highly expressed in half flowering stage. CONCLUSIONS: RNA-seq and DEG data provided comprehensive gene expression information at the transcriptional level that could be facilitate the molecular mechanisms of floral biosynthesis pathways in three developmental phase's flowers in Cymbidium goeringii, moreover providing useful information for further analysis on C. goeringii, and other plants of genus Cymbidium.

Comparative Genomic Analysis for Genetic Variation in Sacbrood Virus of Apis cerana and Apis mellifera Honeybees From Different Regions of Vietnam.

Sacbrood virus (SBV) is one of the most common viral infections of honeybees. The entire genome sequence for nine SBV infecting honeybees, Apis cerana and Apis mellifera, in Vietnam, namely AcSBV-Viet1, AcSBV-Viet2, AcSBV-Viet3, AmSBV-Viet4, AcSBV-Viet5, AmSBV-Viet6, AcSBV-Viet7, AcSBV-Viet8, and AcSBV-Viet9, was determined. These sequences were aligned with seven previously reported complete genome sequences of SBV from other countries, and various genomic regions were compared. The Vietnamese SBVs (VN-SBVs) shared 91-99% identity with each other, and shared 89-94% identity with strains from other countries. The open reading frames (ORFs) of the VN-SBV genomes differed greatly from those of SBVs from other countries, especially in their VP1 sequences. The AmSBV-Viet6 and AcSBV-Viet9 genome encodes 17 more amino acids within this region than the other VN-SBVs. In a phylogenetic analysis, the strains AmSBV-Viet4, AcSBV-Viet2, and AcSBV-Viet3 were clustered in group with AmSBV-UK, AmSBV-Kor21, and AmSBV-Kor19 strains. Whereas, the strains AmSBV-Viet6 and AcSBV-Viet7 clustered separately with the AcSBV strains from Korea and AcSBV-VietSBM2. And the strains AcSBV-Viet8, AcSBV-Viet1, AcSBV-Viet5, and AcSBV-Viet9 clustered with the AcSBV-India, AcSBV-Kor and AcSBV-VietSBM2. In a Simplot graph, the VN-SBVs diverged stronger in their ORF regions than in their 5' or 3' untranslated regions. The VN-SBVs possess genetic characteristics which are more similar to the Asian AcSBV strains than to AmSBV-UK strain. Taken together, our data indicate that host specificity, geographic distance, and viral cross-infections between different bee species may explain the genetic diversity among the VN-SBVs in A. cerana and A. mellifera and other SBV strains.

Homology differences between complete Sacbrood virus genomes from infected Apis mellifera and Apis cerana honeybees in Korea.

Sacbrood virus (SBV) represents a serious threat to the health of managed honeybees. We determined four complete SBV genomic sequences (AmSBV-Kor1, AmSBV-Kor2, AcSBV-Kor3, and AcSBV-Kor4) isolated from Apis mellifera and Apis cerana in various regions of South Korea. A phylogenetic tree was constructed from the complete genomic sequences of these Korean SBVs (KSBVs) and 21 previously reported SBV sequences from other countries. Three KSBVs (not AmSBV-Kor1) clustered with previously reported Korean genomes, but separately from SBV genomes from other countries. The KSBVs shared 90-98 % identity, and 89-97 % identity with the genomes from other countries. AmSBV-Kor1 was least similar (~90 % identity) to the other KSBVs, and was most similar to previously reported strains AmSBV-Kor21 (97 %) and AmSBV-UK (93 %). Phylogenetic analysis of the partial VP1 region sequences indicated that SBVs clustered by host species and country of origin. The KSBVs were aligned with nine previously reported complete SBV genomes and compared. The KSBVs were most different from the other genomes at the end of the 5' untranslated region and in the entire open reading frame. A SimPlot graph of the VP1 region confirmed its high variability, especially between the SBVs infecting A. mellifera and A. cerana. In this genomic region, SBVs from A. mellifera species contain an extra continuous 51-nucleotide sequence relative to the SBVs from A. cerana. This genomic diversity may reflect the adaptation of SBV to specific hosts, viral cross-infections, and the spatial distances separating the KSBVs from other SBVs.

Analysis of the RdRp, intergenic and structural polyprotein regions, and the complete genome sequence of Kashmir bee virus from infected honeybees (Apis mellifera) in Korea.

Kashmir bee virus (KBV) is one of the most common viral infections in honeybees. In this study, a phylogenetic analysis was performed using nine partial nucleotide sequences of RdRp and the structural polyprotein regions of South Korean KBV genotypes, as well as nine previously reported KBV genotypes from various countries and two closely related genotypes of Israeli acute paralysis virus (IAPV) and Acute bee paralysis virus (ABPV). The Korean KBV genotypes were highly conserved with 94-99 % shared identity, but they also shared 88-95 % identity with genotypes from various countries, and they formed a separate KBV cluster in the phylogenetic tree. The complete genome sequence of Korean KBV was also determined and aligned with previously reported complete reference genome sequences of KBV, IAPV, and ABPV to compare different genomic regions. The complete Korean KBV genome shared 93, 79, and 71 % similarity with the complete reference genomes of KBV, IAPV, and ABPV, respectively. The Korean KBV was highly conserved relative to the reference KBV genomes in the intergenic and 3' untranslated region (UTR), but it had a highly variable 5' UTR, whereas there was little divergence in the helicase and 3C-protease of the nonstructural protein, and the external domains of the structural polyprotein region. Thus, genetic recombination and geographical distance may explain the genomic variations between the Korean and reference KBV genotypes.

Analysis of the complete genome sequence and capsid region of black queen cell viruses from infected honeybees (Apis mellifera) in Korea.

Black queen cell virus (BQCV) infection is one of the most common viral infections in honeybees (Apis mellifera). A phylogenetic tree was constructed for 19 partial nucleotide sequences for the capsid region of South Korean BQCV, which were also compared with 10 previously reported BQCV sequences derived from different countries. The Korean BQCV genomes were highly conserved and showed 97-100% identity. They also showed 92-99% similarity with other country genotypes and showed no significant clustering in the phylogenetic tree. In order to investigate this phenomenon in more detail, the complete genome sequence of the Korean BQCV strain was determined and aligned with those from a South African reference strain and European genotypes, Poland4-6 and Hungary10. A phylogenetic tree was then constructed. The Korean BQCV strain showed a high level of similarity (92%) with Hungary10, but low similarity (86%) with the South African reference genotype. Comparison of the Korean and other sequences across different genome regions revealed that the 5'-UTR, the intergenic region, and the capsid regions of the BQCV genome were highly conserved. ORF1 (a non-structural protein coding region) was more variable than ORF2 (a structural protein coding region). The 5'-proximal third of ORF1 was particularly variable and contained several insertions/deletions. This phenomenon may be explained by intra-molecular recombination between the Korean and other BQCV genotypes; this appeared to have happened more with the South African reference strain than with the European genotypes.

Phylogenetic analysis of black queen cell virus genotypes in South Korea.

The black queen cell virus (BQCV), a picorna-like honeybee virus, was first isolated from queen larvae and pupae of honeybees found dead in their cells. BQCV is the most common cause of death in queen larvae. Phylogenetic analysis of two Apis cerana and three Apis mellifera BQCV genotypes collected from honeybee colonies in different regions of South Korea, central European BQCV genotypes, and a South African BQCV reference genotype was performed on a partial helicase enzyme coding region (ORF1) and a partial structural polypeptide coding region (ORF2). The phylogeny based on the ORF2 region showed clustering of all the Korean genotypes corresponding to their geographic origin, with the exception of Korean Am str3 which showed more similarity to the central European and the South African reference genotype. However, the ORF1-based tree exhibited a different distribution of the Korean strains, in which A. cerana isolates formed one cluster and all A. mellifera isolates formed a separate cluster. The RT-PCR assay described in this study is a sensitive and reliable method for the detection and classification of BQCV strains from various regions of Korea. BQCV infection is present in both A. cerana and A. mellifera colonies. With this in mind, the present study examined the transmission of honeybee BQCV infections between A. cerana and A. mellifera.

Progress and Challenges in the Improvement of Ornamental Plants by Genome Editing.

Biotechnological approaches have been used to modify the floral color, size, and fragrance of ornamental plants, as well as to increase disease resistance and vase life. Together with the advancement of whole genome sequencing technologies, new plant breeding techniques have rapidly emerged in recent years. Compared to the early versions of gene editing tools, such as meganucleases (MNs), zinc fingers (ZFNs), and transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) is capable of altering a genome more efficiently and with higher accuracy. Most recently, new CRISPR systems, including base editors and prime editors, confer reduced off-target activity with improved DNA specificity and an expanded targeting scope. However, there are still controversial issues worldwide for the recognition of genome-edited plants, including whether genome-edited plants are genetically modified organisms and require a safety evaluation process. In the current review, we briefly summarize the current progress in gene editing systems and also introduce successful/representative cases of the CRISPR system application for the improvement of ornamental plants with desirable traits. Furthermore, potential challenges and future prospects in the use of genome-editing tools for ornamental plants are also discussed.

Volatiles Profile of the Floral Organs of a New Hybrid Cymbidium, 'Sunny Bell' Using Headspace Solid-Phase Microextraction Gas Chromatography-Mass Spectrometry Analysis.

Cymbidium is one of the most important genera of flowering plants in the Orchidaceae family, and comprises a wide variety of beautiful and colorful species. Among these, only a few species possess floral scents and flavors. In order to increase the availability of a new Cymbidum hybrid, "Sunny Bell", this study investigated the volatile floral scents. Volatiles of the floral organs of the new Cymbidium hybrid, "Sunny Bell", at the full-flowering stage were characterized with headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis. A divinylbenzene-carboxen-polydimethylsiloxane (DVB-CAR-PDMS) fiber gave the best extraction for volatile components. Twenty-three components were identified as the main volatiles for the floral organs of the new Cymbidium hybrid, "Sunny Bell" at the full-flowering stage; twelve compounds in the column, sixteen compounds in the labellum, eleven compounds in the sepals, and nine compounds in the petals were identified. Terpenes are the major source of floral scents in this plant. As a result of GC-MS analysis, the most abundant compound was linalool (69-80%) followed by α-pinene (3-27%), 4,8-dimethyl-1,3,7-nonatriene (5-18%), eucalyptol (6-16%), and 2,6-dimethylnonane (2-16%). The main components were identified as monoterpenes in the petals and sepals, and as monoterpenes and aliphatics in the column and labellum. The results of this study provide a basis for breeding Cymbidium cultivars which exhibit desirable floral scents.

Analysis of the nonstructural and structural polyprotein regions, and complete genome sequences of Israel acute paralysis viruses identified from honeybees (Apis mellifera) in Korea.

Phylogenetic trees were constructed for 24 partial nucleotide sequences of the nonstructural polyprotein (ORF1) and structural polyprotein regions (ORF2) of Korean IAPV genotypes, as well as eight previously reported IAPV sequences from various countries. Most of the Korean genotypes formed a distinct cluster, separate from other country genotypes. To investigate this phenomenon in more detail, three complete IAPV genome sequences were identified from different regions in Korea, i.e., Korea1, Korea2, and Korea3. These sequences were aligned with eight previously reported complete genome sequences and various genome regions were compared. The Korean IAPVs were very similar to those from China and Israel, but highly diverged from USA and Australian genotypes. Interestingly, they showed greater variability than the USA and Australian genotypes in ORF1, but highly similar to the Australian genotype in the ORF2 region. Thus, genetic recombination may account for the spatial distance between the Korean IAPV genotypes and those from other countries.

Molecular characterization and phylogenetic analysis of deformed wing viruses isolated from South Korea.

Deformed wing virus (DWV) is one of the most common viral infection in honeybees. Phylogenetic trees were constructed for 16 partial nucleotide sequences of the structural polyprotein region and the RNA helicase region of South Korean DWVs. The sequences were compared with 10 previously reported DWV sequences from different countries and the sequences of two closely related viruses, Kakugo virus (KGV) and Varroa destructor virus-1 (VDV-1). The phylogeny based on these two regions, the Korean DWV genomes were highly conserved with 95-100% identity, while they also shared 93-97% similarity with genotypes from other countries, although they formed a separate cluster. To investigate this phenomenon in more detail, the complete DWV genome sequences of Korea-1 and Korea-2 were determined and aligned with six previously reported complete DWV genome sequences from different countries, as well as KGV and VDV-1, and a phylogenetic tree was constructed. The two Korean DWVs shared 96.4% similarity. Interestingly, the Korea-2 genome was more similar to the USA (96.5%) genome than the Korea-1. The Korean genotypes highly conserved with USA (96%) but low similarity with the United Kingdom3 (UK3) genome (89%). The end of the 5' untranslated region (UTR), the start of the open reading frame (ORF) region, and the 3' UTR were variable and contained several substitutions/transitions. This phenomenon may be explained by intramolecular recombination between the Korean and other DWV genotypes.