Comparative analyses reveal a phenylalanine ammonia lyase dependent and salicylic acid mediated host resistance in Zingiber zerumbet against the necrotrophic soft rot pathogen Pythium myriotylum.

Little is known about the molecular basis of host defense in resistant wild species Zingiber zerumbet (L.) Smith against the soil-borne, necrotrophic oomycete pathogen Pythium myriotylum Drechsler, which causes the devastating soft rot disease in the spice crop ginger (Zingiber officinale Roscoe). We investigated the pattern of host defense between Z. zerumbet and ginger in response to P. myriotylum inoculation. Analysis of gene expression microarray data revealed enrichment of phenylpropanoid biosynthetic genes, particularly lignin biosynthesis genes, in pathogen-inoculated Z. zerumbet compared to ginger. RT-qPCR analysis showed the robust activation of phenylpropanoid biosynthesis genes in Z. zerumbet, including the core genes PAL, C4H, 4CL, and the monolignol biosynthesis and polymerization genes such as CCR, CAD, C3H, CCoAOMT, F5H, COMT, and LAC. Additionally, Z. zerumbet exhibited the accumulation of the phenolic acids including p-coumaric acid, sinapic acid, and ferulic acid that are characteristic of the cell walls of commelinoid monocots like Zingiberaceae and are involved in cell wall strengthening by cross linking with lignin. Z. zerumbet also had higher total lignin and total phenolics content compared to pathogen-inoculated ginger. Phloroglucinol staining revealed the enhanced fortification of cell walls in Z. zerumbet, specifically in xylem vessels and surrounding cells. The trypan blue staining indicated inhibition of pathogen growth in Z. zerumbet at the first leaf whorl, while ginger showed complete colonization of the pith within 36 h post inoculation (hpi). Accumulation of salicylic acid (SA) and induction of SA regulator NPR1 and the signaling marker PR1 were observed in Z. zerumbet. Silencing of PAL in Z. zerumbet through VIGS suppressed downstream genes, leading to reduced phenylpropanoid accumulation and SA level, resulting in the susceptibility of plants to P. myriotylum. These findings highlight the essential role of PAL-dependent mechanisms in resistance against P. myriotylum in Z. zerumbet. Moreover, our results suggest an unconventional role for SA in mediating host resistance against a necrotroph. Targeting the phenylpropanoid pathway could be a promising strategy for the effective management of P. myriotylum in ginger.

Complete chloroplast genomes provide insights into evolution and phylogeny of Zingiber (Zingiberaceae).

BACKGROUND: The genus Zingiber of the Zingiberaceae is distributed in tropical, subtropical, and in Far East Asia. This genus contains about 100-150 species, with many species valued as important agricultural, medicinal and horticultural resources. However, genomic resources and suitable molecular markers for species identification are currently sparse. RESULTS: We conducted comparative genomics and phylogenetic analyses on Zingiber species. The Zingiber chloroplast genome (size range 162,507-163,711 bp) possess typical quadripartite structures that consist of a large single copy (LSC, 86,986-88,200 bp), a small single copy (SSC, 15,498-15,891 bp) and a pair of inverted repeats (IRs, 29,765-29,934 bp). The genomes contain 113 unique genes, including 79 protein coding genes, 30 tRNA and 4 rRNA genes. The genome structures, gene contents, amino acid frequencies, codon usage patterns, RNA editing sites, simple sequence repeats and long repeats are conservative in the genomes of Zingiber. The analysis of sequence divergence indicates that the following genes undergo positive selection (ccsA, ndhA, ndhB, petD, psbA, psbB, psbC, rbcL, rpl12, rpl20, rpl23, rpl33, rpoC2, rps7, rps12 and ycf3). Eight highly variable regions are identified including seven intergenic regions (petA-pabJ, rbcL-accD, rpl32-trnL-UAG, rps16-trnQ-UUG, trnC-GCA-psbM, psbC-trnS-UGA and ndhF-rpl32) and one genic regions (ycf1). The phylogenetic analysis revealed that the sect. Zingiber was sister to sect. Cryptanthium rather than sect. Pleuranthesis. CONCLUSIONS: This study reports 14 complete chloroplast genomes of Zingiber species. Overall, this study provided a solid backbone phylogeny of Zingiber. The polymorphisms we have uncovered in the sequencing of the genome offer a rare possibility (for Zingiber) of the generation of DNA markers. These results provide a foundation for future studies that seek to understand the molecular evolutionary dynamics or individual population variation in the genus Zingiber.

Phylogenomics and genome size evolution in Amomum s. s. (Zingiberaceae): Comparison of traditional and modern sequencing methods.

BACKGROUND AND AIMS: A targeted enrichment NGS approach was used to construct the phylogeny of Amomum Roxb. (Zingiberaceae). Phylogenies based on hundreds of nuclear genes, the whole plastome and the rDNA cistron were compared with an ITS-based phylogeny. Trends in genome size (GS) evolution were examined, chromosomes were counted and the geographical distribution of phylogenetic lineages was evaluated. METHODS: In total, 92 accessions of 54 species were analysed. ITS was obtained for 79 accessions, 37 accessions were processed with Hyb-Seq and sequences from 449 nuclear genes, the whole cpDNA, and the rDNA cistron were analysed using concatenation, coalescence and supertree approaches. The evolution of absolute GS was analysed in a phylogenetic and geographical context. The chromosome numbers of 12 accessions were counted. KEY RESULTS: Four groups were recognised in all datasets though their mutual relationships differ among datasets. While group A (A. subulatum and A. petaloideum) is basal to the remaining groups in the nuclear gene phylogeny, in the cpDNA topology it is sister to group B (A. repoeense and related species) and, in the ITS topology, it is sister to group D (the Elettariopsis lineage). The former Elettariopsis makes a monophyletic group. There is an increasing trend in GS during evolution. The largest GS values were found in group D in two tetraploid taxa, A. cinnamomeum and A. aff. biphyllum (both 2n = 96 chromosomes). The rest varied in GS (2C = 3.54-8.78 pg) with a constant chromosome number 2n = 48. There is a weak connection between phylogeny, GS and geography in Amomum. CONCLUSIONS: Amomum consists of four groups, and the former Elettariopsis is monophyletic. Species in this group have the largest GS. Two polyploids were found and GS greatly varied in the rest of Amomum.

De novo transcriptome assembly, gene annotation, and EST-SSR marker development of an important medicinal and edible crop, Amomum tsaoko (Zingiberaceae).

BACKGROUND: Amomum tsaoko is a medicinal and food dual-use crop that belongs to the Zingiberaceae family. However, the lack of transcriptomic and genomic information has limited the understanding of the genetic basis of this species. Here, we performed transcriptome sequencing of samples from different A. tsaoko tissues, and identified and characterized the expressed sequence tag-simple sequence repeat (EST-SSR) markers. RESULTS: A total of 58,278,226 high-quality clean reads were obtained and de novo assembled to generate 146,911 unigenes with an N50 length of 2002 bp. A total of 128,174 unigenes were successfully annotated by searching seven protein databases, and 496 unigenes were identified as annotated as putative terpenoid biosynthesis-related genes. Furthermore, a total of 55,590 EST-SSR loci were detected, and 42,333 primer pairs were successfully designed. We randomly selected 80 primer pairs to validate their polymorphism in A. tsaoko; 18 of these primer pairs produced distinct, clear, and reproducible polymorphisms. A total of 98 bands and 96 polymorphic bands were amplified by 18 pairs of EST-SSR primers for the 72 A. tsaoko accessions. The Shannon's information index (I) ranged from 0.477 (AM208) to 1.701 (AM242) with an average of 1.183, and the polymorphism information content (PIC) ranged from 0.223 (AM208) to 0.779 (AM247) with an average of 0.580, indicating that these markers had a high level of polymorphism. Analysis of molecular variance (AMOVA) indicated relatively low genetic differentiation among the six A. tsaoko populations. Cross-species amplification showed that 14 of the 18 EST-SSR primer pairs have transferability between 11 Zingiberaceae species. CONCLUSIONS: Our study is the first to provide transcriptome data of this important medicinal and edible crop, and these newly developed EST-SSR markers are a very efficient tool for germplasm evaluation, genetic diversity, and molecular marker-assisted selection in A. tsaoko.

Research on germplasm diversity of Amomum villosum. Lour in genuine producing area.

BACKGROUND: Genuine Chinese medicine is produced from medicinal plant cultivated in a specific region and is of better quality and efficacy, more consistently qualified and famous than that from the same medicinal plant cultivated in other regions. The cultivating region of genuine medicinal plant is known as the genuine producing area. Yangchun City, which is in Guangdong Province of China, is a genuine producing area for the famous Chinese medicine Amomi Fructus (also called Sharen). Amomi Fructus is the ripe and dry fruit of the Zingiberaceae plant A. villosum Lour.. A. villosum was introduced from the Persian Gulf region and has been cultivated in China for over 1000 years. Until now there are no reports on screening for good germplasm of A. villosum. METHODS: The contents of volatile oil and bornyl acetate of Amomi Fructus from 14 populations were determined with GC method, and the relative contents of the main chemical components in the volatile oils were determined with GC-MS method. Evaluation and variance analysis of the comprehensive quality of the 14 samples were conducted by means of a multi-indicator entropy-weight TOPSIS model (Technique for Order Preference by Similarity to an Ideal Solution) combined with OPLS-DA (Orthogonal Partial Least Squares Discrimination Analysis) and HCA (Hierarchical Clustering Analysis). The ISSR (Inter-Simple Sequence Repeat) molecular marker technique and the UPGMA (unweighted pair-group method with arithmetic means) were employed to analyze the genetic relationship among A. villosum populations. RESULTS: The contents of volatile oil and bornyl acetate differed significantly among the different populations, but the main chemical component in the volatile oil was the same in all the samples, which was bornyl acetate. OPLS-DA results showed that 9 indicators were the main factors influencing the quality differences among the 14 populations. The entropy-weight TOPSIS results showed that there were significant differences in the comprehensive qualities of the 12 populations from the genuine producing area. The best quality of fruit was found in the genuine producing area of Chunwan Town; the qualities of 33% of genuine fruits were lower than that of non-genuine fruits. Twenty-three DNA fragments were obtained by ISSR-PCR amplification using four ISSR primers, eleven of which were polymorphic loci, which accounted for 47.8%. The similarity coefficients (GS) of different populations of A. villosum ranged from 0.6087 to 0.9565. CONCLUSION: There are significant differences among different populations of A. villosum in terms of the kinds of major chemical components and their contents, comprehensive quality and genetic diversity. The germplasm resources of A. villosum are rich in the genuine producing area. It means superior germplasm could be selected in the area. The comprehensive quality of the fruit of A. villosum from the non-genuine producing area is better than some of that from genuine producing area, proving that the non-genuine producing area can also produce Amomi Fructus with excellent quality.

Cytochemical and comparative transcriptome analyses elucidate the formation and ecological adaptation of three types of pollen coat in Zingiberaceae.

BACKGROUND: The pollen ornate surface of flowering plants has long fascinated and puzzled evolutionary biologists for their variety. Each pollen grain is contained within a pollen wall consisting of intine and exine, over which the lipoid pollen coat lies. The cytology and molecular biology of the development of the intine and exine components of the pollen wall are relatively well characterised. However, little is known about the pollen coat, which confers species specificity. We demonstrate three types of pollen coat in Zingiberaceae, a mucilage-like pollen coat and a gum-like pollen coat, along with a pollen coat more typical of angiosperms. The morphological differences between the three types of pollen coat and the related molecular mechanisms of their formation were studied using an integrative approach of cytology, RNA-seq and positive selection analysis. RESULTS: Contrary to the 'typical' pollen coat, in ginger species with a mucilage-like (Caulokaempferia coenobialis, Cco) or gum-like (Hornstedtia hainanensis, Hhn) pollen coat, anther locular fluid was still present at the bicellular pollen (BCP) stage of development. Nevertheless, there were marked differences between these species: there were much lower levels of anther locular fluid in Hhn at the BCP stage and it contained less polysaccharide, but more lipid, than the locular fluid of Cco. The set of specific highly-expressed (SHE) genes in Cco was enriched in the 'polysaccharide metabolic process' annotation term, while 'fatty acid degradation' and 'metabolism of terpenoids and polyketides' were significantly enriched in SHE-Hhn. CONCLUSIONS: Our cytological and comparative transcriptome analysis showed that different types of pollen coat depend on the residual amount and composition of anther locular fluid at the BCP stage. The genes involved in 'polysaccharide metabolism' and 'transport' in the development of a mucilage-like pollen coat and in 'lipid metabolism' and 'transport' in the development of a gum-like pollen coat probably evolved under positive selection in both cases. We suggest that the shift from a typical pollen coat to a gum-like or mucilage-like pollen coat in flowering plants is an adaptation to habitats with high humidity and scarcity of pollinators.

Genome Assembly and Analysis of the Flavonoid and Phenylpropanoid Biosynthetic Pathways in Fingerroot Ginger (Boesenbergia rotunda).

Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.

Diverse function of the PISTILLATA, APETALA 3, and AGAMOUS-like MADS-box genes involved in the floral development in Alpinia hainanensis (Zingiberaceae).

Zingiberaceae is the vital clue and key node in the decreased process of fertile stamens in Zingiberales, helping to understand the evolution of the ginger families. This study focuses on Alpinia hainanensis to investigate the function of B- and C-class MADS-box genes in floral development. The introns size of two B-class genes AhPI and AhAP3, and one C-class gene AhAG are quite variable. By contrast, the positions of the corresponding introns are conserved, resulting in a similar exon size in homologs. The typical region 70 bp-CCAATCA element was not found in the second intron of AhAG compared to AG homologs. The subcellular localization showed that AhAP3 was in both intranuclear and extranuclear. The heterodimer was formed between APETALA3 and PISTILLATA but not between the B- and C-class proteins using Y2H and BiFC. The 35S::AhAG heterologous transformed Arabidopsis had curly and smaller rosette leaves with early flowering. Floral organs had no homeotic conversion, albeit sepals and petals reduced in size. Siliques development was affected and displayed wrinkled and shorter. By contrast, 35S::AhAP3 and 35S::AhPI did not show any modified phenotype in transgenic Arabidopsis thaliana. We first proposed the model for Alpinia flower development. MADS-box transcription factor binding at particular genomic locations and interaction with partners may be crucial for the development of the floral organ.

Quantitative analysis of methoxyflavones discriminates between the two types of Kaempferia parviflora.

INTRODUCTION: Kaempferia parviflora or black ginger is abundantly cultivated because its rhizomes contain methoxyflavones that have many pharmacological properties. K. parviflora can be divided into two types, based on morphological characteristics, but differences in their chemical compositions have never been explored. OBJECTIVES: This research aims to find chemical markers that can be used to differentiate between the two types of K. parviflora, the red-leaf and green-leaf types, by quantifying the amounts of methoxyflavones. MATERIAL AND METHODS: K. parviflora samples were collected from 39 locations in Thailand. Their genetic diversity was assessed by a genotyping-by-sequencing (GBS) technique to construct the population structure. Their chemical compositions were analyzed by high performance liquid chromatography-photodiode array detection to determine the methoxyflavone contents. RESULTS: The population structure based on >3,000 single nucleotide polymorphism (SNP) markers showed that the samples can be divided into two groups, which were consistent with the classification by leaf margin color (red-leaf and green-leaf types). HPLC analysis revealed 3,5,7,3',4'-pentamethoxyflavone (PMF), 5,7-dimethoxyflavone (DMF), 5,7,4'-trimethoxyflavone (TMF), 3,5,7-trimethoxyflavone and 3,5,7,4'-tetramethoxyflavone as major methoxyflavones that can be used as chemical markers. The red-leaf type showed higher amounts of PMF, TMF and 3,5,7,4'-tetramethoxyflavone than the green-leaf type, while the green-leaf type showed higher amounts of DMF and 3,5,7-trimethoxyflavone than the red-leaf type. CONCLUSION: These results provide another approach to discriminate the two types of K. parviflora using chemical profiles alongside genetic and morphological analyses. Therefore, a specific type of K. parviflora can be selected over the other based on preferences for a certain methoxyflavone.

Himalayan orogeny and monsoon intensification explain species diversification in an endemic ginger (Hedychium: Zingiberaceae) from the Indo-Malayan Realm.

The Indo-Malayan Realm is a biogeographic realm that extends from the Indian Subcontinent to the islands of Southeast Asia (Malay Archipelago). Despite being megadiverse, evolutionary hypotheses explaining taxonomic diversity in this region have been rare. Here, we investigate the role of geoclimatic events such as Himalayan orogeny and monsoon intensification in the diversification of the ginger-lilies (Hedychium J.Koenig: Zingiberaceae). We first built a comprehensive, time-calibrated phylogeny of Hedychium with 75% taxonomic and geographic sampling. We found that Hedychium is a very young lineage that originated in Northern Indo-Burma, in the Late Miocene (c. 10.6 Ma). This was followed by a late Neogene and early Quaternary diversification, with multiple dispersal events to Southern Indo-Burma, Himalayas, Peninsular India, and the Malay Archipelago. The most speciose clade IV i.e., the predominantly Indo-Burmese clade also showed a higher diversification rate, suggesting its recent rapid radiation. Our divergence dating and GeoHiSSE results demonstrate that the diversification of Hedychium was shaped by both the intensifications in the Himalayan uplift as well as the Asian monsoon. Ancestral character-state reconstructions identified the occurrence of vegetative dormancy in both clades I and II, whereas the strictly epiphytic growth behavior, island dwarfism, lack of dormancy, and a distinct environmental niche were observed only in the predominantly island clade i.e., clade III. Finally, we show that the occurrence of epiphytism in clade III corresponds with submergence due to sea-level changes, suggesting it to be an adaptive trait. Our study highlights the role of recent geoclimatic events and environmental factors in the diversification of plants within the Indo-Malayan Realm and the need for collaborative work to understand biogeographic patterns within this understudied region. This study opens new perspectives for future biogeographic studies in this region and provides a framework to explain the taxonomic hyperdiversity of the Indo-Malayan Realm.

Molecular evolution of chloroplast genomes in subfamily Zingiberoideae (Zingiberaceae).

BACKGROUND: Zingiberoideae is a large and diverse subfamily of the family Zingiberaceae. Four genera in subfamily Zingiberoideae each possess 50 or more species, including Globba (100), Hedychium (> 80), Kaempferia (50) and Zingiber (150). Despite the agricultural, medicinal and horticultural importance of these species, genomic resources and suitable molecular markers for them are currently sparse. RESULTS: Here, we have sequenced, assembled and analyzed ten complete chloroplast genomes from nine species of subfamily Zingiberoideae: Globba lancangensis, Globba marantina, Globba multiflora, Globba schomburgkii, Globba schomburgkii var. angustata, Hedychium coccineum, Hedychium neocarneum, Kaempferia rotunda 'Red Leaf', Kaempferia rotunda 'Silver Diamonds' and Zingiber recurvatum. These ten chloroplast genomes (size range 162,630-163,968 bp) possess typical quadripartite structures that consist of a large single copy (LSC, 87,172-88,632 bp), a small single copy (SSC, 15,393-15,917 bp) and a pair of inverted repeats (IRs, 29,673-29,833 bp). The genomes contain 111-113 different genes, including 79 protein coding genes, 28-30 tRNAs and 4 rRNA genes. The dynamics of the genome structures, gene contents, amino acid frequencies, codon usage patterns, RNA editing sites, simple sequence repeats and long repeats exhibit similarities, with slight differences observed among the ten genomes. Further comparative analysis of seventeen related Zingiberoideae species, 12 divergent hotspots are identified. Positive selection is observed in 14 protein coding genes, including accD, ccsA, ndhA, ndhB, psbJ, rbcL, rpl20, rpoC1, rpoC2, rps12, rps18, ycf1, ycf2 and ycf4. Phylogenetic analyses, based on the complete chloroplast-derived single-nucleotide polymorphism data, strongly support that Globba, Hedychium, and Curcuma I + "the Kaempferia clade" consisting of Curcuma II, Kaempferia and Zingiber, form a nested evolutionary relationship in subfamily Zingiberoideae. CONCLUSIONS: Our study provides detailed information on ten complete Zingiberoideae chloroplast genomes, representing a valuable resource for future studies that seek to understand the molecular evolutionary dynamics in family Zingiberaceae. The identified divergent hotspots can be used for development of molecular markers for phylogenetic inference and species identification among closely related species within four genera of Globba, Hedychium, Kaempferia and Zingiber in subfamily Zingiberoideae.

Genetic variation and population structure of clonal Zingiber zerumbet at a fine geographic scale: a comparison with two closely related selfing and outcrossing Zingiber species.

BACKGROUND: There has always been controversy over whether clonal plants have lower genetic diversity than plants that reproduce sexually. These conflicts could be attributed to the fact that few studies have taken into account the mating system of sexually reproducing plants and their phylogenetic distance. Moreover, most clonal plants in these previous studies regularly produce sexual progeny. Here, we describe a study examining the levels of genetic diversity and differentiation within and between local populations of fully clonal Zingiber zerumbet at a microgeographical scale and compare the results with data for the closely related selfing Z. corallinum and outcrossing Z. nudicarpum. Such studies could disentangle the phylogenetic and sexually reproducing effect on genetic variation of clonal plants, and thus contribute to an improved understanding in the clonally reproducing effects on genetic diversity and population structure. RESULTS: The results revealed that the level of local population genetic diversity of clonal Z. zerumbet was comparable to that of outcrossing Z. nudicarpum and significantly higher than that of selfing Z. corallinum. However, the level of microgeographic genetic diversity of clonal Z. zerumbet is comparable to that of selfing Z. corallinum and even slightly higher than that of outcrossing Z. nudicarpum. The genetic differentiation among local populations of clonal Z. zerumbet was significantly lower than that of selfing Z. corallinum, but higher than that of outcrossing Z. nudicarpum. A stronger spatial genetic structure appeared within local populations of Z. zerumbet compared with selfing Z. corallinum and outcrossing Z. nudicarpum. CONCLUSIONS: Our study shows that fully clonal plants are able not only to maintain a high level of within-population genetic diversity like outcrossing plants, but can also maintain a high level of microgeographic genetic diversity like selfing plant species, probably due to the accumulation of somatic mutations and absence of a capacity for sexual reproduction. We suggest that conservation strategies for the genetic diversity of clonal and selfing plant species should be focused on the protection of all habitat types, especially fragments within ecosystems, while maintenance of large populations is a key to enhance the genetic diversity of outcrossing species.

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.

Cell-Free Expression of a Plant Membrane Protein BrPT2 From Boesenbergia Rotunda.

Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins.

Development of 23 novel microsatellite markers of Amomum tsao-ko (Zingiberaceae) based on restriction-site-associated DNA sequencing.

Amomum tsao-ko (Zingiberaceae) is a traditional Chinese medicine and condiment, and an important economic crop in the tropical forest of southwest China. However, few simple sequence repeat (SSR) markers are available in A. tsao-ko, which is hindering genetic research in this species. The aim of this study was to develop and characterize microsatellite markers for A. tsao-ko using restriction-site-associated DNA sequencing. A total of 115,482 microsatellites were identified using MISA software, and 13,411 SSR primer pairs were designed. 100 pairs of SSR primers were selected at random and used to evaluate polymorphisms among 4 A. tsao-ko samples. Finally, 23 pairs of SSR primers with clear bands and obvious polymorphism were selected for genetic diversity analysis of 72 A. tsao-ko accessions. The number of alleles and effective number of alleles per locus ranged from 2 to 6 and from 1.315 to 3.776, respectively. The observed heterozygosity ranged from 0.208 to 0.779, and the expected heterozygosity was from 0.239 to 0.735. The average values of the polymorphic information content were 0.454. Hardy-Weinberg equilibrium (HWE) analysis showed that 10 loci significantly deviated from HWE (P < 0.05). The pairwise FST and genetic distance values revealed low levels of genetic differentiation and high genetic similarity among six A. tsao-ko populations. These microsatellite markers developed will provide a valuable tool for further germplasm characterization, genetic diversity, and breeding studies in A. tsao-ko.

Complete chloroplast genomes of Zingiber montanum and Zingiber zerumbet: Genome structure, comparative and phylogenetic analyses.

Zingiber montanum (Z. montanum) and Zingiber zerumbet (Z. zerumbet) are important medicinal and ornamental herbs in the genus Zingiber and family Zingiberaceae. Chloroplast-derived markers are useful for species identification and phylogenetic studies, but further development is warranted for these two Zingiber species. In this study, we report the complete chloroplast genomes of Z. montanum and Z. zerumbet, which had lengths of 164,464 bp and 163,589 bp, respectively. These genomes had typical quadripartite structures with a large single copy (LSC, 87,856-89,161 bp), a small single copy (SSC, 15,803-15,642 bp), and a pair of inverted repeats (IRa and IRb, 29,393-30,449 bp). We identified 111 unique genes in each chloroplast genome, including 79 protein-coding genes, 28 tRNAs and 4 rRNA genes. We analyzed the molecular structures, gene information, amino acid frequencies, codon usage patterns, RNA editing sites, simple sequence repeats (SSRs) and long repeats from the two chloroplast genomes. A comparison of the Z. montanum and Z. zerumbet chloroplast genomes detected 489 single-nucleotide polymorphisms (SNPs) and 172 insertions/deletions (indels). Thirteen highly divergent regions, including ycf1, rps19, rps18-rpl20, accD-psaI, psaC-ndhE, psbA-trnK-UUU, trnfM-CAU-rps14, trnE-UUC-trnT-UGU, ccsA-ndhD, psbC-trnS-UGA, start-psbA, petA-psbJ, and rbcL-accD, were identified and might be useful for future species identification and phylogeny in the genus Zingiber. Positive selection was observed for ATP synthase (atpA and atpB), RNA polymerase (rpoA), small subunit ribosomal protein (rps3) and other protein-coding genes (accD, clpP, ycf1, and ycf2) based on the Ka/Ks ratios. Additionally, chloroplast SNP-based phylogeny analyses found that Zingiber was a monophyletic sister branch to Kaempferia and that chloroplast SNPs could be used to identify Zingiber species. The genome resources in our study provide valuable information for the identification and phylogenetic analysis of the genus Zingiber and family Zingiberaceae.

Comparative studies on population genetic structure of two closely related selfing and outcrossing Zingiber species in Hainan Island.

How mating system impacts the genetic diversity of plants has long fascinated and puzzled evolutionary biologists. Numerous studies have shown that self-fertilising plants have less genetic diversity at both the population and species levels than outcrossers. However, the phylogenetic relationships between species and correlated ecological traits have not been accounted for in these previous studies. Here, we conduct a comparative population genetic study of two closely related selfing and outcrossing Zingiber species, with sympatric distribution in Hainan Island, and obtain a result contrary to previous studies. The results indicate that selfing Z. corallinum can maintain high genetic diversity through differentiation intensified by local adaptation in populations across the species' range. In contrast, outcrossing Z. nudicarpum preserves high genetic diversity through gene exchange by frequent export of pollen within or among populations. Contrary to expectations, the major portion of genetic variation of outcrossing Z. nudicarpum may exist among populations, depending on the dispersal ability of pollen and seed. Our results also reveal that the main factor affecting population structure of selfing Z. corallinum is mountain ranges, followed by a moist climate, while that of outcrossing Z. nudicarpum is likely moisture, but not mountain ranges, due to gene flow via pollen.

Identification and Monitoring of Amomi Fructus and its Adulterants Based on DNA Barcoding Analysis and Designed DNA Markers.

Amomi Fructus is one of the traditional medicines derived from the ripe fruits of the Zingiberaceae family of plants, which include Amomum villosum, A. villosum var. xanthioides, and A. longiligulare. Owing to their highly similar morphological traits, several kinds of adulterants of Amomi Fructus have been reported. Therefore, accurate and reliable methods of identification are necessary in order to ensure drug safety and quality. We performed DNA barcoding using five regions (ITS, matK, rbcL, rpoB, and trnL-F intergenic spacer) of 23 Amomi Fructus samples and 22 adulterants. We designed specific DNA markers for Amomi Fructus based on the single nucleotide polymorphisms (SNPs) in the ITS. Amomi Fructus was well separated from the adulterants and was classified with the species of origin based on the detected SNPs from the DNA barcoding results. The AVF1/ISR DNA marker for A. villosum produced a 270 bases amplified product, while the ALF1/ISF DNA marker produced a 350 bases product specific for A. longiligulare. Using these DNA markers, the monitoring of commercially distributed Amomi Fructus was performed, and the monitoring results were confirmed by ITS analysis. This method identified samples that were from incorrect origins, and a new species of adulterant was also identified. These results confirmed the accuracy and efficiency of the designed DNA markers; this method may be used as an efficient tool for the identification and verification of Amomi Fructus.

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.