Structure,bioavailability and physicochemical properties of icariin-soymilk nanoparticle

Soymilk is a natural nanocarrier.However,the performance of flavonoid-soymilk nano-complex remains unclear.In this work,icariin-soymilk nano-complexes(ISNCs)were successfully fabricated and characterized.The effects of high-pressure homogenization(HPH)treatment on structure and physicochemical properties of soymilk and nano-complexes were investigated.HPH treatment could significantly improve the surface hydrophobicity and interfacial activity of soymilk.The soymilk with HPH treatment could significantly improve the water solubility(20 folds),thermal stability and bioavailability of icariin.The highest encapsulation efficiency(93.28%),loading capacity(39.09μg/mg),ζ-potentia(absolute value,31.20 mV)and bioavailability(72.14%)were observed in HSI-200(200 bar of homogenization pressure).While HSI-500(500 bar of homogenization pressure)showed the smallest particle size(183.73 nm).ISNCs showed a rougher surface and an irregular lamellar structure with large amount of fine particles by using Cryo-SEM,suggesting that icariin was encapsulated in soymilk.These data supplied a novel strategy to improve the performance of icariin in functional foods.

Direct somatic embryogenesis and related gene expression networks in leaf explants of Hippeastrum ‘Bangkok Rose’

Hippeastrum, a highly diverse genus in the Amaryllidaceae family, is a valuable ornamental bulbous flowering plant. Somatic embryogenesis(SE) is an efficient method for mass production of Hippeastrum plantlets. Previous studies have been devoted to the in vitro propagation of Hippeastrum, but the SE and its regulatory networks are rarely reported. In this study, we established a direct SE method of Hippeastrum Bangkok Rose' using leaf bases as explants. MS supplemented with 1.00 mg·L^(-1)NAA +1.00 mg·L^(-1)KT + 0.25 mg·L^(-1)TDZ was the optimal medium for SE. Histological observations showed that the bipolar somatic embryo originated from the epidermal cell layer and underwent initiation,globular, scutellar and coleoptile stages. During SE, endogenous hormones of IAA, CTK, ABA, and SA were highly accumulated. Transcriptomic analysis revealed the genes encoding auxin biosynthesis/metabolic enzymes and efflux carriers were induced, while the auxin receptor of TIR1 and ARF transcriptional repressor of Aux/IAA were down-regulated and up-regulated, respectively, leading to suppression of auxin signaling. In contrast, cytokine signaling was promoted at the early stage of SE, as biosynthesis, transport, and signaling components were up-regulated.Various stress-related genes were up-regulated at the early or late stages of SE. Chromatin remodeling could also be dynamically regulated via distinct expression enzymes that control histone methylation and acetylation during SE. Moreover, key SE regulators, including WOXs and SERKs were highly expressed along with SE. Overall, the present study provides insights into the SE regulatory mechanisms of the Hippeastrum.

Typology and Agroecology of Agroecosystems in Vegetation Dynamics in the Ecotones of the Mbam and Inoubou

Agroforestry systems strongly characterize the Cameroonian agrarian landscape. Agroforests are among those structuring the ecotones of Mbam and Inoubou in the Central Cameroon region. Numerous works on agroecosystems of Central and South Cameroon, few have come out contribution of the structure of these traditional systems in the dynamics of the vegetation of these forest-savannah ecotones. The present contribution has the overall objective of demonstrating the structural efficiency of agroforests the dynamics of ecotone vegetation, but also in the conservation of biodiversity. To do this, a participatory analysis was carried out with 56 farmers distributed in the five villages of Makénéné. Botanical inventories supplemented socio-economic household surveys. The data collected was subjected to various analyzes (univariate test, analysis of variance, multivariate test, PCA, CAH). The results reveal that 55.2% of agroforests are less than 15 years old and those with an area greater than 1500 m2 predominate (33%). They are mainly young with generally small surface areas. The horizontal structure reveals that the largest diameter classes are those of [20 - 30 cm[ and [10 - 20 cm[ with a very low rate of basal area. Agroforests with trees over 10 m high are dominant in five villages of Makénéné. Two types of structural profiles characterize the agroforestry flora of the area, namely intensive pluristratified home gardens on savannah and intensive pluristratified agroforests under forest-savannah transition vegetation. Principal Component Analysis (PCA) and Ascending Hierarchical Clustering (HAC) show three Agroforest Clusters each. The PCA distribution reveals that woody biomass (Y) is strongly correlated with tree diameter (DBH) and significantly with height (Cluster 2). The age (AAG) (Cluster 1) of these agroforests, on the other hand, remains independent of the density (DST) of these trees (Cluster 3). The agroforests in the Nyokon, Carrière and Mocksud villages are the most effective in terms of conserving woody diversity while the agroforests of the Kinding ndé and Nyokon villages are more efficient in the reforestation processes. These results could be considered as effective and quantifiable tools for the certification of numerous cash crops such as cocoa and coffee, which will make it possible to valorize this local knowledge in terms of scientific and in the development of various programs and writing of technical notes.

Functional analysis of flavanone 3-hydroxylase(F3H)from Dendrobium officinale,which confers abiotic stress tolerance

Flavonoids are important bioactive components in Dendrobium officinale,a medicinal orchid.They are involved in many biological activities,including protecting plants against biotic and abiotic stresses.Research on the key genes related to flavonoid biosynthesis in D.officinale is limited.In this study,one of the key flavonoid biosynthesis genes,flavanone 3-hydroxylase(F3H),was characterized from D.officinale.The open reading frame of DoF3H was 1134 bp long and it encoded a 377-amino acid protein.The DoF3H protein showed considerably high homology with F3H proteins from other plant species and shared a common evolutionary ancestor with other F3Hs.DoF3H transcripts were detected in different organs of adult plants and mainly accumulated in flowers,followed by roots,stems and leaves,a pattern that was similar to the content of flavonoids.Recombinant DoF3H protein,which was localized in the cytosol,could convert naringenin to dihydrokaempferol.The mRNA levels of DoF3H were significantly induced by salt and cold stresses.Furthermore,the heterologous expression of DoF3H in Escherichia coli conferred it higher tolerance to salt and cold stresses.These results provide insight into the molecular function of DoF3H in the biosynthesis of flavonoids,and provide a new application for improvement of abiotic tolerance in D.officinale.

Phosphorylation of SiRAV1 at Ser31 regulates the SiCAT expression to enhance salt tolerance in Setaria italica

Salinity severely affects plant growth and development.Thus,it is crucial to identify the genes functioning in salt stress response and unravel the mechanism by which plants against salt stress.This study used the phosphoproteomic assay and found that 123 of the 4000 quantitative analyzed phosphopeptides were induced by salt stress.The functional annotation of the non-redundant protein database(NR)showed 23 differentially expressed transcription factors,including a phosphopeptide covering the Serine 31 in the RAV(related to ABI3/VP1)transcription factor(named SiRAV1).SiRAV1 was located in the nucleus.Phenotypic and physiological analysis showed that overexpressing SiRAV1 in foxtail millet enhanced salt tolerance and alleviated the salt-induced increases of H_(2)O_(2) accumulation,malondialdehyde(MDA)content,and percent of electrolyte leakage.Further analysis showed that SiRAV1 positively regulated SiCAT expression to modulate the catalase(CAT)activity by directly binding to the SiCAT promoter in vivo and in vitro.Moreover,we found that phosphorylation of SiRAV1 at the Ser31 site positively regulated salt tolerance in foxtail millet via enhancing its binding ability to SiCAT promoter but did not affect its subcellular localization.Overall,our results define a mechanism for SiRAV1 function in salt response where salt-triggered phosphorylation of SiRAV1 at Ser31 enhances its binding ability to SiCAT promoter,and the increased SiCAT expression contributes to salt tolerance in foxtail millet.

OsNPF3.1,a nitrate,abscisic acid and gibberellin transporter gene,is essential for rice tillering and nitrogen utilization efficiency

Low-affinity nitrate transporter genes have been identified in subfamilies 4-8 of the rice nitrate transporter 1(NRT1)/peptide transporter family(NPF),but the OsNPF3 subfamily responsible for nitrate and phytohormone transport and rice growth and development remains unknown.In this study,we described OsNPF3.1 as an essential nitrate and phytohormone transporter gene for rice tillering and nitrogen utilization efficiency(NUtE).OsNPF3.1 possesses four major haplotypes of its promoter sequence in 517 cultivars,and its expression is positively associated with tiller number.Its expression was higher in the basal part,culm,and leaf blade than in other parts of the plant,and was strongly induced by nitrate,abscisic acid(ABA)and gibberellin 3(GA_3)in the root and shoot of rice.Electrophysiological experiments demonstrated that OsNPF3.1 is a pH-dependent low-affinity nitrate transporter,with rice protoplast uptake assays showing it to be an ABA and GA_3 transporter.OsNPF3.1 overexpression significantly promoted ABA accumulation in the roots and GA accumulation in the basal part of the plant which inhibited axillary bud outgrowth and rice tillering,especially at high nitrate concentrations.The NUtE of OsNPF3.1-overexpressing plants was enhanced under low and medium nitrate concentrations,whereas the NUtE of OsNPF3.1 clustered regularly interspaced short palindromic repeats(CRISPR)plants was increased under high nitrate concentrations.The results indicate that OsNPF3.1 transports nitrate and phytohormones in different rice tissues under different nitrate concentrations.The altered OsNPF3.1 expression improves NUtE in the OsNPF3.1-overexpressing and CRISPR lines at low and high nitrate concentrations,respectively.

Colonization of Rice Roots by a Green Fluorescent Protein-Tagged Isolate of Ustilaginoidea virens

The fungus U. virens is the causal agent of rice false smut disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Using epifluorescence microscopy colonization and infection on rice roots were visualized in vivo. After inoculation for 2 to 15 d, it was observed that the conidia and their germ-tubes had penetrated into epidermis of young roots. The hyphae were found inside the root xylem 18 d after inoculation. Generally, the transformed fungus colonized the rhizosphere, the cortex as well as the vascular tissues with symptoms of root necrosis observed. The results of this work show that U. virens colonize not only rice panicles but also the roots.

The responses of carbon-and nitrogen-acquiring enzymes to nitrogen and phosphorus additions in two plantations in southern China

In tropical plantations,nutrients such as nitrogen(N)or phosphorus(P)are often applied as management practices.However,the effects of such nutrient additions on topsoil C-and N-acquiring enzymes activities are unclear.In this study,the impacts of fertilization onβ-1,4-glucosidase(BG),β-D-cellobiosidase(CBH),β-1,4-xylosidase(BX),β-1,4-N-acetyl-glucosaminidase(NAG),and leucine amino peptidase(LAP)enzymes activities from topsoil and litter layer of two tropical plantations(Acacia auriculiformis and Eucalyptus urophylla)were measured.The results showed that N addition had neutral impact on topsoil enzymes,while significantly elevating the activities of BG,CBH,BX,and NAG in the litter layer.P fertilization had no impacts except for an elevation of NAG in litter sample.There was no interactions found between N and P additions on these enzyme activities.The clearer impacts of N over P fertilization were unexpected because that the study site receives a high rate of atmospheric N deposition,and has low soil P availability.The impact of P fertilization on hydrolytic enzyme activities may be less important compared with that of N.

Genome size evolution of the extant lycophytes and ferns

Ferns and lycophytes have remarkably large genomes.However,little is known about how their genome size evolved in fern lineages.To explore the origins and evolution of chromosome numbers and genome size in ferns,we used flow cytometry to measure the genomes of 240 species(255 samples)of extant ferns and lycophytes comprising 27 families and 72 genera,of which 228 species(242 samples)represent new reports.We analyzed correlations among genome size,spore size,chromosomal features,phylogeny,and habitat type preference within a phylogenetic framework.We also applied ANOVA and multinomial logistic regression analysis to preference of habitat type and genome size.Using the phylogeny,we conducted ancestral character reconstruction for habitat types and tested whether genome size changes simultaneously with shifts in habitat preference.We found that 2 C values had weak phylogenetic signal,whereas the base number of chromosomes(x)had a strong phylogenetic signal.Furthermore,our analyses revealed a positive correlation between genome size and chromosome traits,indicating that the base number of chromosomes(x),chromosome size,and polyploidization may be primary contributors to genome expansion in ferns and lycophytes.Genome sizes in different habitat types varied significantly and were significantly correlated with habitat types;specifically,multinomial logistic regression indicated that species with larger 2 C values were more likely to be epiphytes.Terrestrial habitat is inferred to be ancestral for both extant ferns and lycophytes,whereas transitions to other habitat types occurred as the major clades emerged.Shifts in habitat types appear be followed by periods of genomic stability.Based on these results,we inferred that habitat type changes and multiple whole-genome duplications have contributed to the formation of large genomes of ferns and their allies during their evolutionary history.

Synthesis and Cytotoxic Activity of Novel Tetrahydrocurcumin Derivatives Bearing Pyrazole Moiety

Tetrahydrocurcumin(THC)is a major metabolite of curcumin and plays an important role in curcumin-induced biological effects.THC is a promising preventive and chemotherapeutic agent for cancer.A series of new pyrazole derivatives of THC have been synthesized as potent anticancer agents.Direct condensation of THC with various substituted hydrazines leads to new pyrazole derivatives of THC(1-18).The prepared compounds have been evaluated via in vitro MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay for their cell proliferation-inhibitory activity against human lung adenocarcinoma(A549),human cervical carcinoma(HeLa)and human breast carcinoma(MCF-7)cells.Most derivatives show significantly higher anticancer activity against all three tested cancer cell lines than the parent compound THC.Several compounds(7,8,12,13 and 15)display promising anticancer activity against MCF-7 cell line with IC50 values ranging from 5.8 to 9.3 iM.The most active compound(8)is substituted with 4-bromophenyl group at the pyrazole ring and inhibits the growth of all three tested cancer cell lines with an IC50 values of(8.0 iM,A549),(9.8 iM,HeLa)and(5.8 iM,MCF-7).The obtained compounds can be a good starting point for the development of new lead molecules in the fight against cancer.

Feasible strategies for studying the involvement of DNA methylation and histone acetylation in the stress-induced formation of quality-related metabolites in tea(Camellia sinensis)

Tea plants are subjected to multiple stresses during growth,development,and postharvest processing,which affects levels of secondary metabolites in leaves and influences tea functional properties and quality.Most studies on secondary metabolism in tea have focused on gene,protein,and metabolite levels,whereas upstream regulatory mechanisms remain unclear.In this review,we exemplify DNA methylation and histone acetylation,summarize the important regulatory effects that epigenetic modifications have on plant secondary metabolism,and discuss feasible research strategies to elucidate the underlying specific epigenetic mechanisms of secondary metabolism regulation in tea.This information will help researchers investigate the epigenetic regulation of secondary metabolism in tea,providing key epigenetic data that can be used for future tea genetic breeding.

Isolation of mesophyll protoplasts from tea(Camellia sinensis)and localization analysis of enzymes involved in the biosynthesis of specialized metabolites

Specialized metabolites in tea(Camellia sinensis)are fundamental quality factors.It is important to characterize gene function in vivo to identify key enzymes and reactions involved in the biosynthesis of such metabolites.Here we report a transient expression method to analyze gene function in isolated tea mesophyll protoplasts.This method is an alternative approach to traditional genetic transformation for studies on gene function in vivo.We screened several tea cultivars and different digestion conditions to optimize protoplast isolation.Digestion of newly emerged leaves of C.sinensis‘Zhongbai 4’with 3%cellulase R-10 and 0.3%macerozyme R-10 for about 12 h yielded approximately 107 mesophyll protoplasts.Genes encoding enzymes involved in secondary metabolite synthesis were transiently expressed in the protoplasts,and their subcellular locations were determined.With further improvements in the transfection efficiency,this transient expression system will contribute greatly to the analyses of in vivo gene function in tea.

Growth Characteristics of <i>Fuirena umbellata</i>in a Surface Flow Constructed Wetland and Its Influence in Nutrients and Faecal Bacteria Removal from Domestic Wastewater in Cameroon

Various configurations of vegetated bed systems with a variety of macrophytes have been tested experimentally in Cameroon, for the treatment of domestic wastewater. The aim of this work was to assess the growth and biomass production of Fuirena umbellata (Cyperaceae) and its potentials in the removal of faecal bacteria and nutrients from primarily treated domestic effluent. A wetland vegetated with this macrophyte and a non-vegetated wetland (control) were continuously fed with primarily treated domestic wastewater at an estimated loading rate of 205 Litres/day in dry and rainy seasons for two consecutive years. Physicochemical and microbiological parameters of the effluent were monitored at the inflow and outflows of the wetlands alongside with the growth and productivity attributes of the young plants during each season. The density of plants ranged in the wetland from 17 - 185 plants/m2 and from 11 - 146 plants/m2 respectively during the first and the second years. More biomasses were instead produced in the dry seasons than in the rainy seasons but with no significant differences observed. As for nutrients removal, higher efficiencies were observed in the vegetated wetland (45% - 73%) compared to the non-vegetated control (17% - 66%). Similar trends were observed for the faecal bacteria but with no significant differences between the seasons. However, the vegetated beds were significantly more efficient than the non-vegetated control in the reduction of many physicochemical parameters and faecal bacteria. This varied with the seasons.

Role of histone deacetylase CsHDA8 in regulating the accumulation of indole during the oolong tea manufacturing process

Aroma is an important quality-related trait of oolong tea.The compounds contributing to oolong tea aromas are mainly produced in response to stress during the enzyme-active stage of the tea manufacturing process.Epigenetic regulation affects stress-induced secondary metabolite production;however,the mechanisms regulating oolong tea aroma compound formation are unclear.In this study,the effect of histone acetylation on indole formation,an important aroma of oolong tea,was revealed and CsHDA8 was identified as a candidate regulator.Additionally,CsHDA8,which was localized in the nucleus and cytoplasm,may function as a histone deacetylase.Moreover,the binding of CsHDA8 to CsTSB2 promoter related to indole decreased during a wounding treatment,which resulted in increased histone acetylation and gene expression.The study results may clarify the regulatory effects of histone acetylation on the formation of tea secondary metabolites,with potential implications for studies on the epigenetic regulation in other non-model horticulture plants.

Plant-Derived Enzymes Producing Chiral Aroma Compounds and Potential Application

Aroma(volatile)compounds play important ecological functions in plants,and also contribute to the quality of plant-derived foods.Moreover,chiral aroma compounds affect their functions in plants and lead to different flavor quality properties.Formations of chiral aroma compounds are due to the presence of enzymes producing these compounds in plants,which are generally involved in the final biosynthetic step of the aroma compounds.Here,we review recent progress in research on the plant-derived enzymes producing chiral aroma compounds,and their changes in response to environmental factors.The chiral aroma enzymes that have been reported produce(R)-linalool,(S)-linalool,(R)-limonene,and(S)-limonene,etc.,and these enzymes are found in various plant species.We also discuss the origins of enantioselectivity in the plant-derived enzymes producing chiral aroma compounds and summarize the potential use of plants containing enzymes producing chiral aroma compounds for producing chiral flavors/fragrances.

The miR5810/OsMRLP6 regulatory module affects rice seedling photosynthesis

Photosynthesis affects crop growth and yield.The roles of microRNAs(miRNAs)in photosynthesis are little known.In the present study,the role of the OsNF-YB7–OsMIR5810–OsMRLP6 regulatory module in photosynthesis was investigated.The malectin-like protein gene OsMRLP6 was identified as a target gene of osa-miR5810(miR5810).Overexpression in rice of miR5810 or down-expression of OsMRLP6 resulted in reduced expression of genes involved in chloroplast development and photosynthesis and decreased net photosynthetic rate,finally leading to lower shoot biomass and grain yield.Down-expression of miR5810 and overexpression of OsMRLP6 showed the opposite effect.Overexpression of transcription factor OsNF-YB7 elevated expression of OsMIR5810 in rice seedlings by binding to its promoter.The OsNFYB7–OsMIR5810–OsMRLP6 regulatory module affects photosynthesis to mediate growth and grain yield.

Peptide Transporter OsNPF8.1 Contributes to Sustainable Growth under Salt and Drought Stresses,and Grain Yield under Nitrogen Deficiency in Rice

Peptide transport is important for plant tissues where rapid proteolysis occurs,especially during germination and senescence,to enhance redistribution of organic nitrogen(N).However,the biological role of peptide transporters is poorly investigated in rice.We characterized the function of the peptide transporter OsNPF8.1 of rice nitrate transporter 1/peptide transporter family(NPF).Ectopic expression of OsNPF8.1 in yeast revealed that OsNPF8.1 encoded a high-affinity di-/tri-peptide transporter,and the osnpf8.1 mutants had a lower uptake rate of the fluorescent-labelled dipeptide c in leaves of rice seedlings.Histochemical assays showed that OsNPF8.1 was highly expressed in mesophyll cells and vascular parenchyma cells,but not detected in root hairs and epidermises.Expression of OsNPF8.1 was induced by N deficiency,drought,Na Cl and abscisic acid,and kept at a high level in senescing leaves.Under N deficiency conditions,compared with the wild type Zhonghua 11,the osnpf8.1 mutants grew slower at the seedling stage,and had lower grain yield and lower N content in the grains.In contrast,OsNPF8.1-over-expressing rice(OsNPF8.1-OE)grew faster at the seedling stage and had a higher grain yield.The osnpf8.1 seedlings were less tolerant to salt and drought stresses.These results suggested that stress-induced organic N transportation mediated by OsNPF8.1 might contribute to balance plant growth and tolerate to salt/drought stress and N-deficiency.

Functions and mechanisms of non-histone protein acetylation in plants^(∞)

Lysine acetylation, an evolutionarily conserved post-translational protein modification, is reversibly catalyzed by lysine acetyltransferases and lysine deacetylases. Lysine acetylation, which was first discovered on histones, mainly functions to configure the structure of chromatin and regulate gene transcriptional activity. Over the past decade, with advances in high-resolution mass spectrometry, a vast and growing number of non-histone proteins modified by acetylation in various plant species have been identified.Lysine acetylation of non-histone proteins is widely involved in regulating biological processes in plants such as photosynthesis, energy metabolism, hormone signal transduction and stress responses. Moreover, in plants, lysine acetylation plays crucial roles in regulating enzyme activity,protein stability, protein interaction and subcellular localization. This review summarizes recent progress in our understanding of the biological functions and mechanisms of non-histone protein acetylation in plants. Research prospects in this field are also noted.

Environment-driven intraspecific variation shows coordination of functional traits of deciduous oaks among and within different biological levels

Deciduous oaks(Quercus spp.)are distributed from subalpine to tropical regions in the northern hemi-sphere and have important roles as carbon sinks and in climate change mitigation.Determining variations in plant functional traits at multiple biological levels and linking them to environmental variables across geographical ranges is important for forecasting range-shifts of broadly-distrib-uted species under climate change.We sampled leaves of five deciduous Quercus spp.covering approximately 20°of latitude(~21°N-41°N)and 20 longitude(~99°E-119°E)across China and measured 12 plant functional traits at different biological levels.The traits varied distinctively,either within each biological level or among different levels driven by climatic and edaphic variables.Traits at the organ level were significantly correlated with those at the cellular and tissue levels,while traits at the whole-plant level only correlated with those at the tissue level.The Quercus species responded to changing environments by regulating stomatal size,leaf thickness and the palisade mesophyll thickness to leaf thickness ratios with contrasting degree of effect to adjust the whole-plant functioning,i.e.,intrinsic water use efficiency(iWUE),carbon supply and nitrogen availability.The results suggest that these deciduous Quercus spp.will maintain vigour by increasing iWUE when subjected to large temperature changes and insufficient moisture,and by accu-mulating leaf non-structural carbohydrates under drought conditions.The findings provide new insights into the inher-ent variation and trait coordination of widely distributed tree species in the context of climate change.

Parahellenia,a new genus segregated from Hellenia(Costaceae)based on phylogenetic and morphological evidence

Previous studies recognized three major lineages of the family Costaceae:a South American clade,an Asian clade and a Costus clade.However,the genus Hellenia within the Asian clade has been shown to be non-monophyletic and its morphology has not been studied carefully.Therefore,the complete plastid genomes of Hellenia species were obtained and the monophyly of Hellenia was tested through four different datasets in this study.Plastid phylogenomic analyses of Costaceae revealed that Hellenia is strongly supported as paraphyletic.Two major clades are recovered,namely the Hellenia s.s.subclade and the Parahellenia subclade.Phylogenetic analyses based on an enlarged taxon sampling of the Asian clade using a two chloroplast markers dataset(trnK intron and trnL-F spacer)confirmed the paraphyly of Hellenia.Meanwhile,morphological analyses suggested that members of the Parahellenia subclade differ from the remaining Hellenia species in many characters including inflorescences,bracts,stigma,axillary buds,floral tubes and labellum.According to the present molecular and morphological evidence,the latter subclade is recognized as a new genus,Parahellenia.Two new species are described,four new combinations are made,and identification keys are also provided.