Ke-Wei Liu

The genome sequence of the orchid Phalaenopsis equestris

Orchidaceae, renowned for its spectacular flowers and other reproductive and ecological adaptations, is one of the most diverse plant families. Here we present the genome sequence of the tropical epiphytic orchid Phalaenopsis equestris, a frequently used parent species for orchid breeding. P. equestris is the first plant with crassulacean acid metabolism (CAM) for which the genome has been sequenced. Our assembled genome contains 29,431 predicted protein-coding genes. We find that contigs likely to be underassembled, owing to heterozygosity, are enriched for genes that might be involved in self-incompatibility pathways. We find evidence for an orchid-specific paleopolyploidy event that preceded the radiation of most orchid clades, and our results suggest that gene duplication might have contributed to the evolution of CAM photosynthesis in P. equestris. Finally, we find expanded and diversified families of MADS-box C/D-class, B-class AP3 and AGL6-class genes, which might contribute to the highly specialized morphology of orchid flowers.

Co-delivery of chemotherapeutic drugs with vitamin E TPGS by porous PLGA nanoparticles for enhanced chemotherapy against multi-drug resistance.

We report a strategy to make use of poly(lactic-co-glycolic acid) nanoparticle (PLGA NPs) for co-delivery of docetaxel (DTX) as a model anticancer drug together with vitamin E TPGS. The latter plays a dual role as a pore-forming agent in the nanoparticles that may result in smaller particle size, higher drug encapsulation efficiency and faster drug release, and also as a bioactive agent that could inhibit P-glycoprotein to overcome multi-drug resistance of the cancer cells, The DTX-loaded PLGA NPs of 0, 10, 20 and 40% TPGS were prepared by the nanoprecipitation method and then characterized for their size and size distribution, surface morphology, physical status and encapsulation efficiency of the drug in the NPs. All four NPs were found of size ranged 100-120 nm and EE ranged 85-95% at drug loading level around 10%. The in vitro evaluation showed that the 48 h IC50 values of the free DTX and the DTX-loaded PLGA NPs of 0, 10, 20% TPGS were 2.619 and 0.474, 0.040, 0.009 μg/mL respectively, which means that the PLGA NPs formulation could be 5.57 fold effective than the free DTX and that the DTX-loaded PLGA NPs of 10 or 20% TPGS further be 11.85 and 52.7 fold effective than the DTX-loaded PLGA NPs of no TPGS (therefore, 66.0 and 284 fold effective than the free DTX). Xenograft tumor model and immunohistological staining analysis further confirmed the advantages of the strategy of co-delivery of anticancer drugs with TPGS by PLGA NPs.

The Dendrobium catenatum Lindl. genome sequence provides insights into polysaccharide synthase, floral development and adaptive evolution

Orchids make up about 10% of all seed plant species, have great economical value, and are of specific scientific interest because of their renowned flowers and ecological adaptations. Here, we report the first draft genome sequence of a lithophytic orchid, Dendrobium catenatum. We predict 28,910 protein-coding genes, and find evidence of a whole genome duplication shared with Phalaenopsis. We observed the expansion of many resistance-related genes, suggesting a powerful immune system responsible for adaptation to a wide range of ecological niches. We also discovered extensive duplication of genes involved in glucomannan synthase activities, likely related to the synthesis of medicinal polysaccharides. Expansion of MADS-box gene clades ANR1, StMADS11, and MIKC*, involved in the regulation of development and growth, suggests that these expansions are associated with the astonishing diversity of plant architecture in the genus Dendrobium. On the contrary, members of the type I MADS box gene family are missing, which might explain the loss of the endospermous seed. The findings reported here will be important for future studies into polysaccharide synthesis, adaptations to diverse environments and flower architecture of Orchidaceae.

C- and D-class MADS-Box Genes from Phalaenopsis equestris (Orchidaceae) Display Functions in Gynostemium and Ovule Development

Gynostemium and ovule development in orchid are unique developmental processes in the plant kingdom. Characterization of C- and D-class MADS-box genes could help reveal the molecular mechanisms underlying gynostemium and ovule development in orchids. In this study, we isolated and characterized a C- and a D-class gene, PeMADS1 and PeMADS7, respectively, from Phalaenopsis equestris. These two genes showed parallel spatial and temporal expression profiles, which suggests their cooperation in gynostemium and ovule development. Furthermore, only PeMADS1 was ectopically expressed in the petals of the gylp (gynostemium-like petal) mutant, whose petals were transformed into gynostemium-like structures. Protein-protein interaction analyses revealed that neither PeMADS1 and PeMADS7 could form a homodimer or a heterodimer. An E-class protein was needed to bridge the interaction between these two proteins. A complementation test revealed that PeMADS1 could rescue the phenotype of the AG mutant. Overexpression of PeMADS7 in Arabidopsis caused typical phenotypes of the D-class gene family. Together, these results indicated that both C-class PeMADS1 and D-class PeMADS7 play important roles in orchid gynostemium and ovule development.

The Apostasia genome and the evolution of orchids

Constituting approximately 10% of flowering plant species, orchids (Orchidaceae) display unique flower morphologies, possess an extraordinary diversity in lifestyle, and have successfully colonized almost every habitat on Earth. Here we report the draft genome sequence of Apostasia shenzhenica, a representative of one of two genera that form a sister lineage to the rest of the Orchidaceae, providing a reference for inferring the genome content and structure of the most recent common ancestor of all extant orchids and improving our understanding of their origins and evolution. In addition, we present transcriptome data for representatives of Vanilloideae, Cypripedioideae and Orchidoideae, and novel third-generation genome data for two species of Epidendroideae, covering all five orchid subfamilies. A. shenzhenica shows clear evidence of a whole-genome duplication, which is shared by all orchids and occurred shortly before their divergence. Comparisons between A. shenzhenica and other orchids and angiosperms also permitted the reconstruction of an ancestral orchid gene toolkit. We identify new gene families, gene family expansions and contractions, and changes within MADS-box gene classes, which control a diverse suite of developmental processes, during orchid evolution. This study sheds new light on the genetic mechanisms underpinning key orchid innovations, including the development of the labellum and gynostemium, pollinia, and seeds without endosperm, as well as the evolution of epiphytism; reveals relationships between the Orchidaceae subfamilies; and helps clarify the evolutionary history of orchids within the angiosperms.

Potent Anti-Tumor Effect Generated by a Novel Human Papillomavirus (HPV) Antagonist Peptide Reactivating the pRb/E2F Pathway

Human papillomavirus type 16 (HPV16) E7 is a viral oncoprotein believed to play a major role in cervical cancer. In this study, an antagonist peptide against HPV16E7 protein was first identified from screening the c7c phage display peptide library. The binding specificity and affinity of the selected peptide to HPV16E7 were tested by competitive enzyme-linked immunosorbent assay (ELISA). The antagonist peptide showed obvious anti-tumor efficacy both in cell lines and animal tumor models. Significant cell proliferation inhibition with high specificity was noted when HPV16-positive cells were treated with the peptide. This anti-tumor efficacy was resulted from overriding the activities of HPV16E7 and reactivating the pRb/E2F pathway, as shown by a series of experiments. Flow cytometry analysis revealed that the selected peptide induced G1 arrest in a dose-dependent manner. Competitive ELISA, pull down, and Co-IP experiments indicated that the selected peptide disrupted the interaction between HPV16E7 and pRb proteins both in vitro and in vivo. Luciferase reporter assay verified that transcription activities of E2F were suppressed by the peptide through restoration of pRb. RT-PCR and Western blot revealed that it reduced cyclins A, D1, and E1 expression, and led to HPV16E7 protein degradation, but pRb protein stabilization. The current study suggests that this specific peptide may serve as a potential therapeutic agent for HPV16-positive cervical cancer.

Conservation ecology of endangered species Paphiopedilum armeniacum (Orchidaceae)

Abstract Paphiopedilum armeniacum is an endangered orchid species, endemic to China. During the period of April 2000 to October 2005, 66 observation sites were selected in Luoshapo of Nushan Mountains in Yunnan, China, to carry out the conservation ecological research on P. armeniacum. A total of 443 genets (1302 ramets in total) of P. armeniacum were sampled, their biological characteristics such as reproductive pattern, phenology, and life cycle were observed, and the ecological habits of the species such as the habitat and the structure of communities were studied. Experiments on ex-situ conservation were conducted, and the cloned ramets were replanted to their original habitat after ex-situ reproduction in Shenzhen, Guangdong, China. The relationships between P. armeniacum and climate, vegetation, other environmental factors in the original habitat, and the biological characteristics of asexual offsprings of P. armeniacum, which were replanted to the original habitat after ex-situ cultivation and reproduction, were investigated. The studies show that P. armeniacum in Luoshapo grows very well in secondary shrub boskets or in tussocks on limestone hills. It has both sexual and asexual reproduction. Asexual reproduction serves to complete the sexual reproduction and to extend the lifetime of genets, while it does not reduce sexual reproduction. There are two modes of asexual reproduction-by tillering or by producing rhizomes. The litter of shrub boskets or tussocks provides P. armeniacum with humus, and the rhizome reproduction of P. armeniacum is an adaptation to the litter-covered condition, i.e. to escape from the unfavorable environment. Blooming rate of ramets is 7.39% ± 1.02%, and fruit set rate from the blooming ramets is 32.23% ± 12.08%. P. armeniacum is able to invade the moderately destroyed forests and those in early restoring but is unable to grow in large dense forests. P. armeniacum also grows very well in artificial spare woods in Shenzhen and can reproduce many cloned ramets, which can normally bloom and yield fruits after being replanted to the original habitat. The results of this study show that P. armeniacum can be conserved by ex-situ conservation and by replanting the ex-situ reproduced ramets to original habitat. On the basis of the analysis of endangered mechanisms of P. armeniacum, it can be concluded that P. armeniacum has strong capability of both asexual and sexual reproductions, and an emergency mechanism consisted of massive production of rhizomes to cope with damage. Because highly effective pollinating insects that facilitate pollination in P. armeniacum are present in the habitat, flowering ramets produce fruits with large quantity of seeds, many of which in turn grow into new genets that can reproduce many cloned ramets. P. armeniacum makes very effective use of its environment and has distinct characteristics of enduring harsh environmental conditions; therefore, rather than its own inherent biological defects, the main threats facing this species are the destruction of its survival space and the wipe-out collecting of the plants as a result of trading. Based on the analysis mentioned above, certain appropriate strategies have been proposed for the conservation of P. armeniacum.

A New Molecular Phylogeny and a New Genus, Pendulorchis, of the Aerides–Vanda Alliance (Orchidaceae: Epidendroideae)

Background The Aerides–Vanda alliance is a complex group in the subtribe Aeridinae (subfamily Epidendroideae, Orchidaceae). Some phylogenetic systems of this alliance have been previously proposed based on molecular and morphological analyses. However, several taxonomic problems within this alliance as well as between it and its allies remain unsolved. Methodology/Principal Findings We utilized ITS and five plastid DNA regions in this phylogenetic analysis. Consensus trees strongly indicate that the Aerides–Vanda alliance is monophyletic, and the 14 genera of this alliance can be grouped into the following clades with 14 subclades: 1. Aerides, comprising two subclades: Rhynchostylis and Aerides; 2. Ascocentropsis; 3. Papilionanthe; 4. Vanda, comprising five subclades: Neofinetia, Christensonia, Seidenfadenia, Ascocentrum, and Vanda–Trudelia, in which Vanda and Trudelia form a subclade; 5. Tsiorchis, comprising three subclades: Chenorchis, Tsiorchis, and two species of Ascocentrum; 6. Paraholcoglossum; and 7. Holcoglossum. Among the 14 genera, only Ascocentrum is triphyletic: two species of the Ascocentrum subclade, an independent subclade Ascocentrum subclade in the Tsiorchis clade; the Ascocentrum subclade in the Vanda clade; and one species in the Holcoglossum clade. The Vanda and Trudelia species belong to the same subclade. The molecular conclusion is consistent with their morphological characteristics. Conclusions We elucidate the relationship among the 14 genera of the Aerides–Vanda alliance. Our phylogenetic results reveal that the Aerides–Vanda alliance is monophyletic, but it can be divided into 14 genera. The data prove that Ascocentrum is triphyletic. Plants with elongate-terete leaves and small flowers should be treated as a new genus, Pendulorchis. Saccolabium himalaicum (Ascocentrum himalaicum) should be transferred to Pendulorchis. Ascocentrum pumilum, endemic to Taiwan, should be transferred to Holcoglossum. A new combination, Holcoglossum pumilum, was also established. Trudelia should not be recognized as an independent genus. Two new species, Pendulorchis gaoligongensis and Holcoglossum singchianum, were described as well.

A floral organ moving like a caterpillar for pollinating

Abstractu2003 A new pollination mechanism is reported of an orchid species, Bulbophyllum penicillium, based on a field observation in Southeast Yunnan of China. This species has a sensitive lip, and there is a distance of 2–3 mm between it and column apex. Once the lip is touched by a landing insect, it will move up and down or swing left and right continuously, just like a moving caterpillar. By suck a lip movement, the pollinator, a very small fruit fly (Drosophila sp.) ca. 1 mm in height, will be pressed toward the column apex where anther and stigma are located, and then cross‐pollination takes place. This unique mode of pollination, depending on the movement of lip rather than insect itself, has never been found before in either Orchidaceae or other families of angiosperms.

A new orchid genus, Danxiaorchis, and phylogenetic analysis of the tribe Calypsoeae.

Background Orchids have numerous species, and their speciation rates are presumed to be exceptionally high, suggesting that orchids are continuously and actively evolving. The wide diversity of orchids has attracted the interest of evolutionary biologists. In this study, a new orchid was discovered on Danxia Mountain in Guangdong, China. However, the phylogenetic clarification of this new orchid requires further molecular, morphological, and phytogeographic analyses. Methodology/Principal Findings A new orchid possesses a labellum with a large Y-shaped callus and two sacs at the base, and cylindrical, fleshy seeds, which make it distinct from all known orchid genera. Phylogenetic methods were applied to a matrix of morphological and molecular characters based on the fragments of the nuclear internal transcribed spacer, chloroplast matK, and rbcL genes of Orchidaceae (74 genera) and Calypsoeae (13 genera). The strict consensus Bayesian inference phylogram strongly supports the division of the Calypsoeae alliance (not including Dactylostalix and Ephippianthus) into seven clades with 11 genera. The sequence data of each species and the morphological characters of each genus were combined into a single dataset. The inferred Bayesian phylogram supports the division of the 13 genera of Calypsoeae into four clades with 13 subclades (genera). Based on the results of our phylogenetic analyses, Calypsoeae, under which the new orchid is classified, represents an independent lineage in the Epidendroideae subfamily. Conclusions Analyses of the combined datasets using Bayesian methods revealed strong evidence that Calypsoeae is a monophyletic tribe consisting of eight well-supported clades with 13 subclades (genera), which are all in agreement with the phytogeography of Calypsoeae. The Danxia orchid represents an independent lineage under the tribe Calypsoeae of the subfamily Epidendroideae. This lineage should be treated as a new genus, which we have named Danxiaorchis, that is parallel to Yoania. Both genera are placed under the subtribe Yoaniinae.