Transcriptome and hormone metabolome reveal the mechanism of stem bending in water lily (Nymphaea tetragona) cut-flowers.

Water lilies are popular ornamental cut-flowers with significant economic and cultural value. However, stem bending affects the preservation of cut-flowers during their vase life. To gain further insights into the molecular mechanisms of stem bending, transcriptome profiling, hormone measurement, and morphological analysis were performed using the stems of the 'Blue Bird' water lily. Transcriptome analysis revealed that 607 differentially expressed genes (DEGs) were associated with the dorsal and ventral stems of the water lily, of which 247 were up-regulated and 360 were down-regulated. Significant differences in genes associated with plant hormones, calcium ions, glucose metabolism, and photosynthesis pathways genes involved in the dorsal and ventral areas of the curved stem. In particular, DEGs were associated with the hormone synthesis, gravity response, starch granules, Ca2+ ions, and photosynthesis. The results of qRT-PCR were consistent with that of the transcriptome sequence analysis. A total of 12 hormones were detected, of which abscisic acid, indole-3-carboxaldehyde, indole-3-carboxaldehyde and jasmonic acid were significantly differentially expressed in the dorsal and ventral stems, and were significantly higher in the dorsal stem than in the ventral stem. The cell morphology in the dorsal and ventral areas of the curved stem clearly changed during vase life. The direction of starch granule settlement was consistent with the bending direction of the water lily stem, as well as the direction of gravity. In conclusion, stem bending in water lily cut-flowers is regulated by multiple factors and genes. This study provides an important theoretical basis for understanding the complex regulatory mechanism of water lily stem bending.

A new lithophilous species of Gesneriaceae, Petrocodonrubrostriatus, from the karst area of South Yunnan, China.

A new lithophytic species of Gesneriaceae, Petrocodonrubrostriatus K.Tan, X.Q.Song & M.X.Ren, sp. nov. from Lvchun County, South Yunnan, China, is described and illustrated here. It closest resembles P.mollifolius (W.T.Wang) A.Weber & Mich.Möller, but the new species is differentiated from it by red to brownish-red stripes in the yellow corolla throat and 4.5 mm long bract lobes, a ca. 10 mm long style, and staminodes inserted at 2.5-3 mm from the corolla base. The species is preliminarily assessed as 'Critically Endangered' (CR) according to IUCN criteria, since currently only one single locality is known with a few subpopulations on a fragmented limestone cliff, with fewer than 300 individuals.

Comparison of Composition, Free-Radical-Scavenging Capacity, and Antibiosis of Fresh and Dry Leave Aqueous Extract from Michelia shiluensis.

Numerous plants of medicinal value grow on Hainan Island (China). Given the lack of knowledge on the phytochemical and pharmacological properties of Michelia shiluensis Chun and Y. F. Wu (M. shiluensis), the application of natural antioxidants and antimicrobials in the food industry has attracted increasing interest. This study aimed to compare the chemical composition, free-radical-scavenging capacity, and antibiosis of aqueous extracts of the fresh and dried leaves of M. shiluensis. The aqueous extract of the leaves of M. shiluensis was obtained using steam distillation, and its chemical components were separated and identified via gas chromatography-mass spectrometry (GC-MS). The free-radical-scavenging capacity and antibiosis were determined. Further, 28 and 20 compounds were isolated from the fresh leaf aqueous extract of M. shiluensis (MSFLAE) and dried leaf aqueous extract of M. shiluensis (MSDLAE), respectively. The free-radical-scavenging capacity of MSFLAE and MSDLAE was determined by the 2,2-diphenyl-1 picrylhydrazyl (DPPH) method, which was 43.43% and 38.74%, respectively. The scavenging capacity of MSFLAE and MSDLAE determined by the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS)) method was 46.90% and 25.99%, respectively. The iron ion reduction capacity of MSFLAE and MSDLAE was determined by the ferric-reducing antioxidant power (FRAP) method as 94.7 and 62.9 µmol Fe2⁺/L, respectively. This indicated that the two leaf aqueous extracts had a certain free-radical-scavenging capacity, and the capacity of MSFLAE was higher than that of MSDLAE. The antibiosis of the two leaf aqueous extracts on the three foodborne pathogenic bacteria was low, but the antimicrobial effects on Gram-positive bacteria were better than those on Gram-negative bacteria. The antibiosis of MSFLAE on Escherichia coli and Staphylococcus aureus was greater than that of MSDLAE. Finally, MSFLAE and MSDLAE both had certain free-radical-scavenging capacities and antibiosis, confirming that the use of this plant in the research and development of natural antioxidants and antibacterial agents was reasonable. Plant aqueous extracts are an essential source of related phytochemistry and have immense pharmacological potential.

Gastrodiabawanglingensis (Orchidaceae, Epidendroideae), a new species from Hainan Island, China.

Gastrodiabawanglingensis, a new species of Orchidaceae from Hainan Island, China, is described and illustrated. It is morphologically similar to G.theana, G.albidoides and G.albida with dwarf habits, scarcely opening flowers, elongated fruit stems, curved and fleshy perianth tubes and similar columns and lips, but can be easily distinguished from them by having a pair of lateral wings bent outwards at the apex of the column and lateral wings with acuminate tips lower than the anther. According to the IUCN Red List Categories and Criteria, the new species is assessed as Endangered (EN). The plastome of G.bawanglingensis is greatly reduced and reconfigured with approximately 30876 bp in size and 25.36% in GC content. Morphological characteristics and molecular phylogenetic results based on chloroplast gene sequences support the recognition of G.bawanglingensis as a new species within Gastrodia.

DhMYB2 and DhbHLH1 regulates anthocyanin accumulation via activation of late biosynthesis genes in Phalaenopsis-type Dendrobium.

Phalaenopsis-type Dendrobium is a popular orchid with good ornamental and market value. Despite their popularity, molecular regulation of anthocyanin biosynthesis during flower development remains poorly understood. In this study, we systematically investigated the regulatory roles of the transcription factors DhMYB2 and DhbHLH1 in anthocyanins biosynthesis. Gene expression analyses indicated that both DhMYB2 and DhbHLH1 are specifically expressed in flowers and have similar expression patterns, showing high expression in purple floral tissues with anthocyanin accumulation. Transcriptomic analyses showed 29 differentially expressed genes corresponding to eight enzymes in anthocyanin biosynthesis pathway have similar expression patterns to DhMYB2 and DhbHLH1, with higher expression in the purple lips than the yellow petals and sepals of Dendrobium 'Suriya Gold'. Further gene expression analyses and Pearson correlation matrix analyses of Dendrobium hybrid progenies revealed expression profiles of DhMYB2 and DhbHLH1 were positively correlated with the structural genes DhF3'H1, DhF3'5'H2, DhDFR, DhANS, and DhGT4. Yeast one-hybrid and dual-luciferase reporter assays revealed DhMYB2 and DhbHLH1 can bind to promoter regions of DhF3'H1, DhF3'5'H2, DhDFR, DhANS and DhGT4, suggesting a role as transcriptional activators. These results provide new evidence of the molecular mechanisms of DhMYB2 and DhbHLH1 in anthocyanin biosynthesis in Phalaenopsis-type Dendrobium.

Transcriptome Analysis Revealing the Interaction of Abscisic Acid and Cell Wall Modifications during the Flower Opening and Closing Process of Nymphaea lotus.

As a tropical flower, Nymphaea lotus is a typical night-blooming waterlily used in water gardening. Its petals are rich in aromatic substances that can be used to extract essential oils and as flower tea. However, the short life of the flower seriously affects the development of its cut flowers. At present, neither the mechanism behind the night-opening waterlily flower's opening and closing nor the difference between day-opening and night-opening waterlily flowers' opening and closing mechanisms are clear. In this study, endogenous hormone contents of closed (CP) and open (OP) petals were measured, and transcriptome analysis of CP and OP petals was carried out to determine the signal transduction pathway and metabolic pathway that affect flower opening and closing. ABA and cell wall modification were selected as the most significant factors regulating flowering. We used qRT-PCR to identify the genes involved in the regulation of flower opening in waterlilies. Finally, by comparing the related pathways with those of the diurnal type, the obvious difference between them was found to be their hormonal regulation pathways. In conclusion, the endogenous ABA hormone may interact with the cell wall modification pathway to induce the flowering of N. lotus. Our data provide a new direction for the discovery of key factors regulating the flower opening and closing of N. lotus and provide basic theoretical guidance for future horticultural applications.

Morpho-histology, endogenous hormone dynamics, and transcriptome profiling in Dacrydium pectinatum during female cone development.

Dacrydium pectinatum de Laubenfels is a perennial dioeciously gymnosperm species dominant in tropical montane rain forests. Due to deforestation, natural disasters, long infancy, and poor natural regeneration ability, the population of this species has been significantly reduced and listed as an endangered protected plant. To better understand the female cone development in D. pectinatum, we examined the morphological and anatomical changes, analyzed the endogenous hormone dynamics, and profiled gene expression. The female reproductive structures were first observed in January. The morpho-histological observations suggest that the development of the D. pectinatum megaspore can be largely divided into six stages: early flower bud differentiation, bract primordium differentiation, ovule primordium differentiation, dormancy, ovule maturity, and seed maturity. The levels of gibberellins (GA), auxin (IAA), abscisic acid (ABA), and cytokinin (CTK) fluctuate during the process of female cone development. The female cones of D. pectinatum need to maintain a low level of GA3-IAA-ABA steady state to promote seed germination. The first transcriptome database for female D. pectinatum was generated, revealing 310,621 unigenes. Differential expression analyses revealed several floral (MADS2, AGL62, and LFY) and hormone biosynthesis and signal transduction (CKX, KO, KAO, ABA4, ACO, etc.) genes that could be critical for female cone development. Our study provides new insights into the cone development in D. pectinatum and the foundation for female cone induction with hormones.

Full-length transcriptome revealed the accumulation of polyunsaturated fatty acids in developing seeds of Plukenetia volubilis.

Background: Plukenetia volubilis is cultivated as a valuable oilseed crop, and its mature seeds are rich in polyunsaturated fatty acids (FAs), which are widely used in food and pharmaceutical industries. Recently, next-generation sequencing (NGS) transcriptome studies in P. volubilis indicated that some candidate genes were involved in oil biosynthesis. The NGS were inaccuracies in assembly of some candidate genes, leading to unknown errors in date analyses. However, single molecular real-time (SMRT) sequencing can overcome these assembled errors. Unfortunately, this technique has not been reported in P. volubilis. Methods: The total oil content of P. volubilis seed (PVS) was determined using Soxhlet extraction system. The FA composition were analyzed by gas chromatography. Combining PacBio SMRT and Illumina technologies, the transcriptome analysis of developing PVS was performed. Functional annotation and differential expression were performed by BLAST software (version 2.2.26) and RSEM software (version 1.2.31), respectively. The lncRNA-targeted transcripts were predicted in developing PVS using LncTar tool. Results: By Soxhlet extraction system, the oil content of superior plant-type (SPT) was 13.47% higher than that of inferior plant-type (IPT) at mature PVS. The most abundant FAs were C18:2 and C18:3, among which C18:3 content of SPT was 1.11-fold higher than that of IPT. Combined with PacBio and Illumina platform, 68,971 non-redundant genes were obtained, among which 7,823 long non-coding RNAs (lncRNAs) and 7,798 lncRNA-targeted genes were predicted. In developing seed, the expressions of 57 TFs showed a significantly positive correlation with oil contents, including WRI1-like1, LEC1-like1, and MYB44-like. Comparative analysis of expression profiles between SPT and IPT implied that orthologs of FAD3, PDCT, PDAT, and DAGT2 were possibly important for the accumulation of polyunsaturated FAs. Together, these results provide a reference for oil biosynthesis of P. volubilis and genetic improvement of oil plants.

Characterization of the Key Bibenzyl Synthase in Dendrobium sinense.

Dendrobium sinense, an endemic medicinal herb in Hainan Island, is rich in bibenzyls. However, the key rate-limited enzyme involved in bibenzyl biosynthesis has yet to be identified in D. sinense. In this study, to explore whether there is a significant difference between the D. sinense tissues, the total contents of bibenzyls were determined in roots, pseudobulbs, and leaves. The results indicated that roots had higher bibenzyl content than pseudobulbs and leaves. Subsequently, transcriptomic sequencings were conducted to excavate the genes encoding type III polyketide synthase (PKS). A total of six D. sinense PKS (DsPKS) genes were identified according to gene function annotation. Phylogenetic analysis classified the type III DsPKS genes into three groups. Importantly, the c93636.graph_c0 was clustered into bibenzyl synthase (BBS) group, named as D. sinense BBS (DsBBS). The expression analysis by FPKM and RT-qPCR indicated that DsBBS showed the highest expression levels in roots, displaying a positive correlation with bibenzyl contents in different tissues. Thus, the recombinant DsBBS-HisTag protein was constructed and expressed to study its catalytic activity. The molecular weight of the recombinant protein was verified to be approximately 45 kDa. Enzyme activity analysis indicated that the recombinant DsBBS-HisTag protein could use 4-coumaryol-CoA and malonyl-CoA as substrates for resveratrol production in vitro. The Vmax of the recombinant protein for the resveratrol production was 0.88 ± 0.07 pmol s-1 mg-1. These results improve our understanding with respect to the process of bibenzyl biosynthesis in D. sinense.

Metabolite analysis in Nymphaea 'Blue Bird' petals reveal the roles of flavonoids in color formation, stress amelioration, and bee orientation.

In this study, metabolome of open petals (OP) and closed petals (CP) from Nymphaea 'Blue Bird' was firstly investigated. A total of 455 metabolites was identified in Nymphaea 'Blue Bird' petals, which was mainly composed of 100 flavonoids, 83 phenolic acids, 64 amino acids and derivatives, 60 lipids, 32 alkaloids, 32 organic acids, 24 nucleotides and derivatives, and 12 lignans and coumarins. By differential analysis, 192 metabolites were identified with variable importance in project ≥ 1, among which 83 and 109 metabolites were up- and down-regulated in OP group, respectively. Further analysis (Log2 fold change ≥ 1) identified 26 and 7 metabolites exhibited significantly lower and higher contents in CP group, relative to OP group. Importantly, KEGG analysis indicated that flavonoid biosynthesis exhibited the most significant enrichment. qRT-PCR analysis indicated that the PAL, CHS, and HCDBR genes showed a significantly higher expression in OP group than in CP group. These data explain the increase of naringenin chalcone and phloretin in OP. However, there was no significant difference of total flavonoids between OP and CP groups. Considering the increase of H2O2 content and ultraviolet (UV) absorption peak in OP, our results implied that diurnal stressful conditions induced the degradation of flavonoids, which contributed to environmental stress amelioration. Moreover, a higher absorption peak of 360-380 nm UV was observed in the extract liquor of OP. The sensitivity maximum of the UV-photoreceptor of bees is situated around 340-380 nm UV. This suggested, as noted for the maximum absorption of dihydrokaempferol in 340-370 nm, rhythmic accumulation and loss of these differential flavonoids in Nymphaea 'Blue Bird' petals might enhance UV pattern to some degree, influencing pollinator attraction.

Transcriptome Analysis Reveals the Senescence Process Controlling the Flower Opening and Closure Rhythm in the Waterlilies (Nymphaea L.).

Most waterlily flowers open at dawn and close after noon usually for three to four days, and thereafter wilt. The short lifespan of flowers restricts the development of the flower postharvest industry. The termination of flower movements is a key event during flower aging process. However, it is still unclear when the senescence process initiates and how it terminates the movement rhythm. In this study, we observed that the opening diameter of flowers was the smallest on the fourth (last) flowering day. Subsequent transcriptome profiles generated from petals at different flowering stages showed that the multiple signaling pathways were activated at the last closure stage (Time 3, T3) of the flowers, including Ca2+, reactive oxygen species and far red light signaling pathways, as well as auxin, ethylene and jasmonic acid signaling pathways. Moreover, In terms of cell metabolism regulation, the genes related to hydrolase (protease, phospholipase, nuclease) were upregulated at T3 stage, indicating that petals entered the senescence stage at that time; and the genes related to water transport and cell wall modification were also differentially regulated at T3 stage, which would affect the ability of cell expand and contract, and eventually lead to petal not open after the fourth day. Collectively, our data provided a new insight into the termination of flower opening in the waterlilies, and a global understanding of the senescence process of those opening-closure rhythm flowers.

Genome-Wide Identification and Expression Analysis of MYB Transcription Factor Superfamily in Dendrobium catenatum.

Dendrobium catenatum is an important traditional Chinese medicine and naturally grows on tree trunks and cliffs, where it can encounter diverse environmental stimuli. MYB transcription factors are widely involved in response to abiotic stresses. However, the MYB gene family has not yet been systematically cataloged in D. catenatum. In this study, a total of 133 MYB proteins were identified in D. catenatum, including 32 MYB-related, 99 R2R3-MYB, 1 3R-MYB, and 1 4R-MYB proteins. Phylogenetic relationships, conserved motifs, gene structures, and expression profiles in response to abiotic stresses were then analyzed. Phylogenetic analysis revealed MYB proteins in D. catenatum could be divided into 14 subgroups, which was supported by the conserved motif compositions and gene structures. Differential DcMYB gene expression and specific responses were analyzed under drought, heat, cold, and salt stresses using RNA-seq and validated by qRT-PCR. Forty-two MYB genes were differentially screened following exposure to abiotic stresses. Five, 12, 11, and 14 genes were specifically expressed in response to drought, heat, cold, and salt stress, respectively. This study identified candidate MYB genes with possible roles in abiotic tolerance and established a theoretical foundation for molecular breeding of D. catenatum.

Transcriptomics and Metabolomics Reveal Purine and Phenylpropanoid Metabolism Response to Drought Stress in Dendrobium sinense, an Endemic Orchid Species in Hainan Island.

Drought stress is a bottleneck factor for plant growth and development, especially in epiphytic orchids that absorb moisture mainly from the air. Recent studies have suggested that there are complex transcriptional regulatory networks related to drought stress in Dendrobium sinense. In this study, the transcription and metabolite alterations involved in drought stress response in D. sinense were investigated through RNA-seq and metabolomics. A total of 856 metabolites were identified from stressed and control samples, with 391 metabolites showing significant differences. With PacBio and Illumina RNA sequencing, 72,969 genes were obtained with a mean length of 2,486 bp, and 622 differentially expressed genes (DEGs) were identified. Correlation analysis showed 7 differential genes, and 39 differential metabolites were involved in interaction networks. The network analysis of differential genes and metabolites suggested that the pathways of purine metabolism and phenylpropanoid biosynthesis may play an important role in drought response in D. sinense. These results provide new insights and reference data for culturally important medicinal plants and the protection of endangered orchids.

First Report of Lasiodiplodia theobromae and L. pseudotheobromae Causing Canker Disease of Sacha Inchi in Hainan, China.

Sacha inchi (Plukenetia volubilis L.) belongs to the family Euphorbiaceae. It is a perennial wooden oilseed crop, and also exhibits a good source of polyunsaturated fatty acids, protein and other bioactive compounds, such as tocopherols, carotenes and phytosterols (Chirinos et al. 2013). During 2017-2018 survey, canker disease showing greyish-brown sunken lesions was observed on the branches of sacha inchi in Danzhou campus, Hainan University, China. The disease incidence is less than 5%. However, it can lead to leaf yellowing, wilt, and eventually the whole plant death. In Nov. 2017, twelve branches showing the typical canker symptoms were collected and covered with parafilm at both ends of all samples to prevent desiccation and placed in black plastic bags keeping at 4°C until isolations were made. Samples were rinsed with tap water and dried with paper towels. Fragments, 5mm in length and cut from the junction of diseased and healthy parts of branches, were surface-sterilized with 2% sodium hypochlorite solution for 2 min, rinsed with sterilized distilled water for 5 times, dried by sterilized filter paper, plated on PDA medium amended with 100 µg/mL streptomycin (PDA-str) and incubated in the dark for 4 days at 28°C. Pure cultures of fungal isolates were obtained by transferring mycelial fragments from colony margins onto fresh PDA plates and incubated as described before. The colonies of cultures were initially white, and eventually turned black after 4 days on PDA medium (Fig S1A). The morphology characterization of conidia produced by the isolates was initially hyaline and aseptate (Fig S1B), and a single median septum formed in the mature conidia (Fig S1B). The average size of 50 conidia was 16.39±1.46ⅹ 8.52±0.92µm for J6, and 15.64±1.73ⅹ 8.94±0.86µm for J3. Three genes were used for phylogenetic analysis (Alves et al. 2006). ITS regions and the partial of TUB (ß-tubulin gene) were amplified using the primer pairs ITS1 and ITS4 (White et al. 1990), Bt2a and Bt2b (Glass and Donaldson 1995), respectively, and EF1-688F/EF1-1251R for J3 and EF1-728F/EF1-986R for J6 were used to amplify TEF (translation elongation factor 1-alpha) (Alves et al. 2008). The sequences of ITS, TUB and TEF from J3 and J6 were deposited in Gene-Bank (Table S1). The blast searches in Gene-Bank with ITS, TUB and TEF amplified from isolates J3, respectively, revealed 100, 99, and 100% identities with L. pseudotheobromae, and isolate J6 showed 100, 100 and 99% of identity with L. theobromae. The phylogenetic analysis of the combined ITS, TUB and TEF sequences of J3, J6 and 28 reference strains retrieved from Gene-Bank was performed using the program MEGA 6.0 evaluated by 1000 bootstrap replications, and the result was consistent with the conclusion above (Fig S2). With the phylogenic studies supported by morphological characters, J3 was identified as L. pseudotheobromae and J6 was L. theobromae. For the pathogenicity test, J3 and J6 were used to inoculate 4-week-old healthy sacha inchi potted seedlings. One wound about 5 mm in depth per seedling stem was made using a sterile blade. A 5-mm-diameter mycelium plug of each isolate taken from the edge of 4-day-old culture growing on PDA was placed to the freshly wound of each plant stem and the inoculated area was wrapped with Parafilm. Sterile PDA plugs were placed onto the wounds of control seedlings. Nine healthy seedlings were inoculated with each isolate or PDA plugs in a completely randomized design. After inoculation, plants were placed in a greenhouse at room temperature (26 to 30°C, 80% RH) and were irrigated when needed. The experiment was conducted twice. Five days later, black or dark-brown canker lesions formed on the stems of inoculated plants, and expended upward and downward from the inoculation points. Pycnidia produced on the necrotic regions and were used to to observe the morphology of conidia (Fig S3). The fungus L. pseudotheobromae or L. theobromae can be re-isolated from the inoculated plants, but not from the control ones. L. pseudotheobromae was recorded to be collected from dead leaves of P. volubilis in Yunnan Province, China, but did not prove this fungus to be pathogenic (Tennakoon et al. 2016). This is the first report that L. theobromae and L. pseudotheobromae causing stem canker in sacha inchi in Hainan, China. The results pave the way for the development of management strategies for canker disease in sacha inchi.

Genetic delimitation of Oreocharis species from Hainan Island.

Hainan Island harbours an extraordinary diversity of Gesneriaceae with 14 genera and 23 species, amongst which two species and one variety are recognised in the genus Oreocharis. These three Oreocharis taxa are all Hainan-endemics and show a complex geographical distribution pattern with considerable morphological intermixtures. In this study, we combined DNA (nuclear ITS sequences and cpDNAtrnL-trnF and ycf1b) to evaluate genetic delimitation for 12 Oreocharis populations from the island, together with morphological similarity analysis using 16 morphological traits. The results showed Hainan Oreocharis taxa were monophyletic with relative low genetic diversity within populations, highly significant genetic differentiation amongst populations and a significant phylogeographical structure. The 12 populations formed three genetically distinct groups, roughly correspondent to the currently recognised two species and one unknown lineage. The PCA analyses of morphological traits indicate three distinctive groups, differing mainly in petal colour and corolla shapes. The roles of river and mountain isolations in the origin and distribution of these three lineages are discussed.

Complete plastome sequence of Wrightia laevis Hook. f. a dyestuff species.

Wrightia laevis Hook. f. is a great tree of Apocynaceae. It is mainly distributed in Southeast provinces of China and Southeast Asian countries. It is a plant that combines dyestuff and economic value. There is no study on the genome of W. laevisso far. Here we report and characterize the complete plastid genome sequence of W. laevis in order to provide genomic resources useful for promoting its conservation. The complete chloroplast genome of W. laevis is 155,274 bp in length with a typical quadripartite structure, consisting of a large single-copy region (LSC, 85,463 bp), a single-copy region (SSC, 18,181 bp) and a pair of inverted repeats (IRs, 25,815 bp). There are 133 genes annotated, including 88 unique protein-coding genes, 8 unique ribosomal RNA genes, and 37 transfer RNA genes. The overall G/C content in the plastome of W. laevis is 38.05%. The complete plastome sequence of W. laevis will provide a useful resource for the conservation genetics of this species as well as for phylogenetic studies in Apocynaceae.

Complete plastome sequence of Caesalpinia sappan Linnaeus, a dyestuff and medicinal species.

Caesalpinia sappan Linnaeus is a great tree of Fabaceae. It is mainly distributed in the Southern provinces of China and Southeast Asian countries. It can be used to extract dyes. The heartwood has dyestuff and medicinal value. There is no study on the genome of C. sappan so far. Here, we report and characterize the complete plastid genome sequence of C. sappan in an order to provide genomic resources useful for promoting its conservation. The complete chloroplast genome of C. sappan is 160,176 bp in length with a typical quadripartite structure, consisting of a large single-copy region (LSC, 89,710 bp), a single-copy region (SSC, 18,357 bp), and a pair of inverted repeats (IRs, 26,054 bp). There are 129 genes annotated, including 84 unique protein-coding genes, eight unique ribosomal RNA genes, and 37 transfer RNA genes. The overall G/C content in the plastome of C. sappan is 36.0%. The complete plastome sequence of C. sappan will provide a useful resource for the conservation genetics of this species as well as for phylogenetic studies in Apocynaceae.

mRNA and miRNA Expression Analysis Reveal the Regulation for Flower Spot Patterning in Phalaenopsis 'Panda'.

Phalaenopsis cultivar 'Panda' is a beautiful and valuable ornamental for its big flower and unique big spots on the petals and sepals. Although anthocyanins are known as the main pigments responsible for flower colors in Phalaenopsis, and the anthocyanins biosynthetic pathway in Phalaenopsis is generally well known, the detailed knowledge of anthocynins regulation within the spot and non-spot parts in 'Panda' flower is limited. In this study, transcriptome and small RNA libraries analysis from spot and non-spot sepal tissues of 'Panda' were performed, and we found PeMYB7, PeMYB11, and miR156g, miR858 is associated with the purple spot patterning in its sepals. Transcriptome analyses showed a total 674 differentially expressed genes (DEGs), with 424 downregulated and 250 upregulated (Non-spot-VS-Spot), and 10 candidate DEGs involved in anthocyanin biosynthetic pathway. The qPCR analysis confirmed that seven candidate structure genes (PeANS, PeF3'H, PeC4H, PeF3H, PeF3H1, Pe4CL2, and PeCHI) have significantly higher expressing levels in spot tissues than non-spot tissues. A total 1552 differentially expressed miRNAs (DEMs) were detected with 676 downregulated and 876 upregulated. However, microRNA data showed no DEMs targeting on anthocyanin biosynthesis structure gene, while a total 40 DEMs target transcription factor (TF) genes, which expressed significantly different level in spot via non-spot sepal, including 2 key MYB regulator genes. These results indicated that the lack of anthocyanidins in non-spot sepal may not directly be caused by microRNA suppressing anthocyanidin synthesis genes rather than the MYB genes. Our findings will help in understanding the role of miRNA molecular mechanisms in the spot formation pattern of Phalaenopsis, and would be useful to provide a reference to similar research in other species.

Ultrasound-Enhanced Subcritical Fluid Extraction of Essential Oil from Nymphaea alba var and Its Antioxidant Activity.

Background: The essential oil content of the water lily is extremely low; thus, finding a new method that can extract essential oil from water lilies with a high extraction rate and no residual organic solvents is essential. Objective: The optimal processing conditions for the ultrasound-enhanced subcritical fluid extraction of essential oil from Nymphaea alba var (red water lily) and the antioxidant activity of the essential oil in vitro are investigated to provide theoretical bases for identification and development. Methods: Single-factor experiments and orthogonal designs are performed to determine the effects of extraction conditions on essential oil yields. The chemical composition of essential oil is analyzed using GC-MS. Results: The optimum extraction parameters are established as follows: extraction temperature, 35°C; extraction time, 30 min/time for four times; ratio of material to liquid, 1:3; ultrasound power, 250 W/L; and ultrasonic frequency, 20 kHz. The extraction rate of essential oil is 0.315% under these conditions. Eleven components comprise more than 1% content. The main chemical constituents are 8-hexadecyne (31.04%) and 2,6,10-trimethyl-tetradecane (3.95%). The essential oil from N. alba var has an antioxidant activity in vitro; however, its antioxidant activity is weaker than that of butylated hydroxytoluene. Conclusions: Subcritical fluid is suitable for the extraction of essential oil from N. alba var, and the essential oil has a good antioxidant activity. Highlights: The essential oil content of N. alba var is 0.315%. Forty-seven chemical constituents are identified and isolated from N. alba var and analyzed by GC-MS.

Chemical constituents from Dendrobium hainanense.

A new phenolic derivative (1) and a new dihydrophenanthrene (2) were isolated from the aerial part of Dendrobium hainanense rofe, along with 12 known compounds. The structures of the new compounds were elucidated by spectroscopic analysis，and the relative configuration of compound 1 was determined by J-based configuration analysis (JBCA) method. Bioassay result indicated that compound 1 exhibited weak antibacterial activity against Canidia albicans and Ralstonia solanaceanum.