High effects of climate oscillations on population diversity and structure of endangered Myricaria laxiflora.

Exploring the effects of climate oscillations on the population diversity and structure of endangered organisms in the Three Gorges Reservoir (TGR) area is essential for hydrological environment changes on endangered organism evolution. Myricaria laxiflora is an endemic and endangered shrub restricted to the TGR along the banks of Yangtze River, China. Recently, six natural populations of this species were newly found upstream and downstream of the TGR, whose habitats have been dramatically changed by the summer flooding regulated by large dams. To study the water level fluctuations and climatic shifts on the genetic diversity and genetic differentiation of the six natural populations, 303 individuals from six populations were analyzed based on one nuclear DNA (ITS) and four chloroplast fragments (trnL-F, psbA-trnH, rps16, and rpl16). The phylogenetic tree and significant genetic divergence identified in the cpDNA and ITS with genetic isolation and limited gene flow among regions suggested that the six populations separated well to two groups distributed upstream and downstream. The MaxEnt modeling results indicated that obvious unidirectional eastward migration via Yangtze River gorges watercourse mediated from Last Interglacial to Last Glacial Maximum were showed with the narrow scale distributions of six remnant populations and nine extirpated populations. The initial habitat fragmentation could be triggered by the accumulation of local habitat loss of the impoundment of the TGR during the Present period and might remain stable restoration with bidirectional diffusion in the Future. Divergences among M. laxiflora populations might have been induced by the drastic changes of the external environment and limited seed/pollen dispersal capacity, as the results of long-term ecological adaptability of summer flooding stress. The haplotypes of nuclear gene could be used for population's differentiation and germplasm protection. This identified gene flow and range dynamics have provided support for the gene-flow and geology hypothesis. It is also crucial for rescuing conservation to understand the impact of environmental dynamics on endangered organism evolution.

The complete plastid genome of Rhamnus leptacantha Schneid. (Rhamnaceae).

Rhamnus leptacantha C.K.Schneid. (1914). is a rare shrub species of the genus Rhamnus. The complete plastid genome of Rhamnus leptacantha was sequenced for the first time in this study. The total length of this genome is 161,248 bp with a large single copy (LSC) region (89,386 bp), a small single copy (SSC) region (19,000 bp), and two inverted repeat regions (IRs, 26,431 bp). A total of 133 functional genes were annotated, including 88 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Plastome of R. leptacantha displayed a conservative structure and gene order. Phylogenetic analysis strongly supported R. leptacantha clustered with other members of genus Rhamnus. This study provides a foundation for further investigation of the complete chloroplast genome in inferring the evolution within the Rhamnaceae family.

The complete chloroplast genome of Goodyera yunnanensis Schltr.

The family Orchidaceae is renowned for its extensive diversity. Within this family, the genus Goodyera R. Br. is classified under the subtribe Goodyerinae, comprising approximately 99 species. In this study, a species Goodyera yunnanensis Schltr., its plastid genome was characterized. The plastid genome of G. yunnanensis is 146,197 bp in size and exhibits a typical quadripartite structure with a pair of inverted repeat regions (IRs) of 25,611 bp, a large single-copy region (LSC) of 81,300 bp and a small single-copy region (SSC) of 13,675 bp. A total of 126 genes were identified, containing 80 protein-coding genes, 38 tRNA genes and 8 rRNA genes. The overall GC content is 37.2%, with corresponding values of 43.3%, 34.7% and 29.1% in IR, LSC and SSC regions, respectively. Forty-seven simple sequence repeats (SSRs) are found in G. yunnanensis plastome, and the frequency of mononucleotide repeats is significantly higher than other repeat types. Phylogenetic analysis indicates that Goodyera is resolved into four clades. G. yunnanensis belongs to the monophyletic clade A, and its phylogenetic position can be reasonably supported by morphological and molecular data.

Summer dormancy of Myricaria laxiflora to escape flooding stress: Changes in phytohormones and enzymes induced by environmental factors.

Dormancy is an adaptation mechanism of plants to environmental stress. Myricaria laxiflora undergoes a long period of flooding stress every year. In order to determine whether this species escapes flooding stress through dormancy, young branches and leaves were collected at different time points before the onset of flooding, and changes in the content/activity of hormones/enzymes that are closely involved in plant growth were monitored. The inducing environmental factors of summer dormancy were identified. The branches and leaves of M. laxiflora showed the following trends as summer flooding approached: (1) gradual increase in the abscisic acid content; (2) gradual decrease in the gibberellin and cytokinin contents; and (3) a continuous decrease in the activities of malate dehydrogenase (MDH), ribulose diphosphate carboxylase (RuBisCo), and glycolate oxidase (GLO). Pearson correlation analysis revealed (1) daylight duration was highly correlated with the hormone content and enzyme activity; (2) the daily mean air temperature (DMAT) was significantly correlated with the cytokinin content. These findings suggest that daylight duration was the main environmental factor leading to changes in the phytohormone content and enzyme activity as well as leading to summer dormancy. M. laxiflora undergoes dormancy before the onset of summer flooding to escape summer flooding stress. Our data indicate that summer flooding does not impede the survival and growth of M. laxiflora.

Responses of leaf functional traits to different hydrological regimes and leaf economics spectrum in the water level fluctuation zone of Three Gorges Reservoir, China.

A unique riparian ecosystem has been created as a result of anti-seasonal flooding after reservoir operations, which notably influences the distribution patterns of plant communities and their functional characteristics in the riparian zone. Plant functional traits which reflect the physiological and ecological processes of plants in particular ecosystems are crucial for indicating the variations in the ecosystem structure and function. To better understand the adaptation strategies of plants to hydrological changes and provide a scientific basis for the selection of species in the re-vegetation of the newly formed ecosystems, 14 leaf functional traits and leaf economics spectrum (LES) of 19 dominant plants under different hydrological conditions were investigated in the water level fluctuation zone (WLFZ) of the Three Gorges Reservoir (TGR). The results showed that anti-seasonal flooding has significant effects on the leaf functional traits of plants (P < 0.05). The net photosynthetic rate of annual plants was significantly higher than that of perennial plants (P < 0.05), and there was a significant correlation between leaf phenotypic and photosynthetic traits (P < 0.05). Canonical correspondence analysis showed that soil water content and available phosphorus were the main factors affecting the leaf function of dominant species, indicating that hydrologic factors were still important environmental factors affecting leaf functional traits of dominant species in the WLFZ. And annuals from the WLFZ have characteristics of thick leaves, high photosynthetic rate, short lifespan, and high nutrient concentrations, which make them close to the fast investment-return end of LES. On the contrary, perennials are close to the slow investment-return end of LES. The high productivity investment of annuals is better than the high defense investment of perennials for adapting to the special habitats in the WLFZ. These results indicated that different functional plants in the WLFZ of the TGR under different hydrological regimes can adopt different strategies by weighing the associations and trade-offs between their economic traits.

Integrated Transcriptomic and Proteomic Analyses Uncover the Regulatory Mechanisms of Myricaria laxiflora Under Flooding Stress.

Flooding is one of the major environmental stresses that severely influence plant survival and development. However, the regulatory mechanisms underlying flooding stress remain largely unknown in Myricaria laxiflora, an endangered plant mainly distributed in the flood zone of the Yangtze River, China. In this work, transcriptome and proteome were performed in parallel in roots of M. laxiflora during nine time-points under the flooding and post-flooding recovery treatments. Overall, highly dynamic and stage-specific expression profiles of genes/proteins were observed during flooding and post-flooding recovery treatment. Genes related to auxin, cell wall, calcium signaling, and MAP kinase signaling were greatly down-regulated exclusively at the transcriptomic level during the early stages of flooding. Glycolysis and major CHO metabolism genes, which were regulated at the transcriptomic and/or proteomic levels with low expression correlations, mainly functioned during the late stages of flooding. Genes involved in reactive oxygen species (ROS) scavenging, mitochondrial metabolism, and development were also regulated exclusively at the transcriptomic level, but their expression levels were highly up-regulated upon post-flooding recovery. Moreover, the comprehensive expression profiles of genes/proteins related to redox, hormones, and transcriptional factors were also investigated. Finally, the regulatory networks of M. laxiflora in response to flooding and post-flooding recovery were discussed. The findings deepen our understanding of the molecular mechanisms of flooding stress and shed light on the genes and pathways for the preservation of M. laxiflora and other endangered plants in the flood zone.

Genome-Wide Characterization and Expression Analysis of HD-ZIP Gene Family in Dendrobium officinale.

The homeodomain-leucine zipper (HD-ZIP) gene family, as one of the plant-specific transcription factor families, plays an important role in regulating plant growth and development as well as in response to diverse stresses. Although it has been extensively characterized in many plants, the HD-ZIP family is not well-studied in Dendrobium officinale, a valuable ornamental and traditional Chinese medicinal herb. In this study, 37 HD-ZIP genes were identified in Dendrobium officinale (Dohdzs) through the in silico genome search method, and they were classified into four subfamilies based on phylogenetic analysis. Exon-intron structure and conserved protein domain analyses further supported the prediction with the same group sharing similar gene and protein structures. Furthermore, their expression patterns were investigated in nine various tissues and under cold stress based on RNA-seq datasets to obtain the tissue-specific and cold-responsive candidates. Finally, Dohdz5, Dohdz9, and Dohdz12 were selected to validate their expression through qRT-PCR analysis, and they displayed significantly differential expression under sudden chilling stress, suggesting they might be the key candidates underlying cold stress response. These findings will contribute to better understanding of the regulatory roles of the HD-ZIP family playing in cold stress and also will provide the vital targets for further functional studies of HD-ZIP genes in D. officinale.

Genome survey of Misgurnus anguillicaudatus to identify genomic information, simple sequence repeat (SSR) markers, and mitochondrial genome.

BACKGROUND: The dojo loach Misgurnus anguillicaudatus is an important economic species in Asia because of its nutritional value and broad environmental adaptability. Despite its economic importance, genomic data for M. anguillicaudatus is currently unavailable. METHODS AND RESULTS: In the present study, we conducted a genome survey of M. anguillicaudatus using next-generation sequencing technology. Its genome size was estimated to be 1105.97 Mb by using K-mer analysis, and its heterozygosity ratio, repeat sequence content, GC content were 1.45%, 58.98%, and 38.03%, respectively. A total of 376,357 microsatellite motifs were identified, and mononucleotides, with a frequency of 42.57%, were the most frequently repeated motifs, followed by 40.83% dinucleotide, 7.49% trinucleotide, 8.09% tetranucleotide, and 0.91% pentanucleotide motifs. The AC/GT, AAT/ATT, and ACAG/CTGT repeats were the most abundant motifs among dinucleotide, trinucleotide, and tetranucleotide motifs, respectively. Besides, the complete mitochondrial genome was sequenced. Based on the Maximum Likelihood and Bayesian inference analyses, M. anguillicaudatus yingde in this study was the "introgressed" mitochondrial type. Seventy microsatellite loci were randomly selected from detected SSR loci to test polymorphic, of which, 20 microsatellite loci were assessed in 30 individuals from a wild population. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) per locus ranged from 7 to 19, 0.400 to 0.933, and 0.752 to 0.938, respectively. All 20 loci were highly informative (PIC > 0.700). Eight loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.05). CONCLUSIONS: This is the first report of genome survey sequencing in M. anguillicaudatus, genome information, mitochondrial genome, and microsatellite markers will be valuable for further studies on population genetic analysis, natural resource conservation, and molecular marker-assisted selective breeding.

Youngiahangii (Asteraceae, Crepidinae), a new species from Hubei, China.

Youngiahangii T.Deng, D.G.Zhang, Qun Liu & Z.M.Li, sp. nov., a new species of Asteraceae, is described and illustrated. It was collected in Wufeng County, Hubei Province, Eastern Central China. Youngiahangii is morphologically most similar to Y.rubida, but can be easily distinguished from the latter by capitula with 8-10 florets and the hairy leaf surface. Phylogenetic analyses, based on the internal transcribed spacers (ITS) and one chloroplast marker (rps16), showed that Y.hangii and Y.rubida were sister species with good support. The results of both phylogenetic analysis and the morphological data support the specific rank of Y.hangii.

Morphological structures and histochemistry of roots and shoots in Myricaria laxiflora (Tamaricaceae).

Myricaria laxiflora (Tamaricaceae) is an endangered plant that is narrowly distributed in the riparian zone of the Three Gorges, along the Yangtze River, China. Using bright-field and epifluorescence microscopy, we investigated the anatomical and histochemical features that allow this species to tolerate both submerged and terrestrial environments. The adventitious roots of Myr. laxiflora had an endodermis with Casparian bands and suberin lamellae; the cortex and hypodermal walls had lignified thickenings in the primary structure. In the mature roots, the secondary structure had cork. The apoplastic barriers in stems consisted of a lignified fiber ring and a cuticle at the young stage and cork at the mature stage. The leaves had two layers of palisade tissue, a hyaline epidermis, sunken stomata, and a thick, papillose cuticle. Aerenchyma presented in the roots and shoots. Several Myr. laxiflora structures, including aerenchyma, apoplastic barriers in the roots and shoots, were adapted to riparian habitats. In addition, shoots had typical xerophyte features, including small leaves, bilayer palisade tissues, sunken stomata, a thick, papillose cuticle, and a hyaline epidermis. Thus, our study identified several anatomical features that may permit Myr. laxiflora to thrive in the riparian zone of the Three Gorges, China.

R-spondin1 in loach (Misgurnus anguillicaudatus): Identification, characterization, and analysis of its expression patterns and DNA methylation in response to high-temperature stress.

With a well-understood function in mammals, R-spondin1 (Rspo1) is an important regulator of ovarian development via the Wnt/ß-catenin pathway. Rspo1 deficiency causes retardation of ovarian development in XX fish, and increases Rspo1 function induces femininity and sex reversal in XY fish. In this study, Rspo1 was successfully cloned from loach (Misgurnus anguillicaudatus), and its expression profile was analyzed. The full-length cDNA of Misgurnus anguillicaudatus Rspo1 (MaRspo1) comprised 1322 bp and included an open reading frame (ORF) of 795 bp, which encoded a predicted polypeptide measuring 264 amino acids in length. Phylogenetic and gene structure analyses showed a highly conserved sequence of MaRspo1 (identical to the Rspo1 genes of other species), consisting of an N-terminal signal peptide (SP), two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat (TSP1) and a C-terminal region. Real-time PCR revealed the female-biased expression profile of MaRspo1, with the highest expression level among tested tissues detected in ovary. Investigation of MaRspo1 expression levels throughout the early development stage (10-60 days post hatching) under three temperature treatments (25 °C, 28 °C, and 31 °C) revealed significantly differential expression of MaRspo1 among the three temperature groups, with decreased MaRspo1 expression in the high-temperature (31 °C) group. The results of DNA methylation analysis indicated that exposure to high temperature during early development can increase the average promoter methylation level of MaRspo1 in both females and males. Taken together, the results of this study provide the basis for the further investigation of the molecular mechanism of Rspo1 in response to temperature.

Apoplastic histochemical features of plant root walls that may facilitate ion uptake and retention.

We used brightfield and epifluorescence microscopy, as well as permeability tests, to investigate the apoplastic histochemical features of plant roots associated with ion hyperaccumulation, invasion, and tolerance of oligotrophic conditions. In hyperaccumulator species with a hypodermis (exodermis absent), ions penetrated the root apex, including the root cap. By contrast, in non-hyperaccumulator species possessing an exodermis, ions did not penetrate the root cap. In vivo, the lignified hypodermis blocked the entry of ions into the cortex, while root exodermis absorbed ions and restricted them to the cortex. The roots of the hyperaccumulators Pteris vittata and Cardamine hupingshanensis, as well as the aquatic invasives Alternanthera philoxeroides, Eichhornia crassipes, and Pistia stratiotes, contained lignin and pectins. These compounds may trap and store ions before hypodermis maturation, facilitating ion hyperaccumulation and retention in the apoplastic spaces of the roots. These apoplastic histochemical features were consistent with certain species-specific characters, including ion hyperaccumulation, invasive behaviors in aquatic environments, or tolerance of oligotrophic conditions. We suggest that apoplastic histochemical features of the root may act as invasion mechanisms, allowing these invasive aquatic plants to outcompete indigenous plants for ions.

Morphological and anatomical adaptations to dry, shady environments in Adiantum reniforme var. sinense (Pteridaceae).

The natural distribution of the rare perennial fern Adiantum reniforme var. sinense (Pteridaceae), which is endemic to shady cliff environments, is limited to small areas of Wanzhou County, Chongqing, China. In this study, we used brightfield and epifluorescence microscopy to investigate the anatomical structures and histochemical features that may allow this species to thrive in shady, dry cliff environments. The A. reniforme var. sinense sporophyte had a primary structure and a dictyostele. The plants of this species had an endodermis, sclerenchyma layers and hypodermal sterome, reflecting an adaption to dry cliff environments. Blades had a thin cuticle and isolateral mesophyll, suggesting a tolerance of shady environments. These characteristics are similar to many sciophyte ferns such as Lygodium japonicum and Pteris multifida. Thus, the morphological and anatomical characteristics of A. reniforme var. sinense identified in this study are consistent with adaptations to shady, dry cliff environments.

Ultrasound-assisted transdermal delivery of alendronate for the treatment of osteoporosis.

The aim of the current research work was to develop sonophoresis-assisted transdermal patches for the treatment of osteoporosis. In the present investigation, we formulated alendronate-chitosan nanoparticles by ionotropic external gelation method. The prepared nanoparticles were found to be smooth and free-flowing. The optimized formulation showed 82.7% of drug release over a period of 12 hours with 99.54% EE, the particle size of 250 nm, PDI 0.22 and zeta potential of 28 mV. The solvent casting evaporation method was used for the development of the patches using HPMC as rate-controlling polymer and dibutyl phthalate as the plasticizer. The optimized patch formulation was found acceptable in terms of physical characteristics (appearance, thickness, folding endurance, weight variation, moisture loss and uptake). The drug content was found to be 99.66±0.9 % with 69.44% of drug permeation through the rat skin. The TP3 formulation had drug content of 99.96% which was the highest among all of the formulations and showed relatively controlled skin permeation of 69.44% over the period of 12 hours. Nearly six-time enhancement of bioavailability was observed when alendronate was used in the nanoparticulate form in transdermal patches used with sonophoresis. Over the period of seven days, the plasma calcium concentration in the rat model was decreased from 16 mg/dl to 4 mg/dl (4 times) in rat groups treated with the transdermal patches containing CS-ALN-NP while the concentration dropped only to 12 mg/dl in case of the transdermal patches containing pure Alendronate. These findings (enhanced skin permeation, enhanced bioavailability and suppression of the plasma calcium level) regarding the transdermal delivery system suggest a promising approach for the treatment of osteoporosis.

Assessment of fracture healing properties of lovastatin loaded nanoparticles: preclinical study in rat model.

Bone fracture, being mainly caused by mechanical stress, requires special and quick attention for a rapid healing. The study presented here aims at formulating nanoparticulate system to overcome the solubility issues of lovastatin. The lovastatin nanoparticles were successfully prepared by ionotropic gelation method using chitosan and tri-polyphosphate as polymers. Thus prepared nanoparticles were found to be smooth and spherical with average particle size of 87 nm and encapsulation efficiency of 86.5%. The in-vitro drug release was found to be almost 89.6% in the first 360 minutes. Artificial fracture was produced in female Wistar rats at right leg using fracture apparatus. After administration of lovastatin nanoparticles or saline solution, the respective groups were observed for various parameters. The X-ray imaging showed that lovastatin accelerated bone healing, compared to control. The growth of animals was not hampered by lovastatin by any means. The radiographic examination confirmed a role of lovastatin in increasing bone density. The histological study showed the broken, proliferated and discontinued trabecullae in the control, while at the same time point, the normal, thick, continuous and connected trabecullae were observed in animals administered with lovastatin nanoparticles. The biomechanical studies showed high breaking resilience and minimum bone brittleness in animals injected with lovastatin nanoparticles. Considering these observations we state that lovastatin helps in rapid bone healing after fracture via increasing the bone density.

[MUC1-2VNTR DNA Vaccine Induces Immune Responses in Mouse Model with Multiple Myeloma].

OBJECTIVE: To investigate the humoral and cellular immune responses induced by MUC1-2VNTR DNA vaccine in multiple myeloma (MM) tumor-bearing mice. METHODS: In vitro, multiple myeloma cells were transfected by plasmid pcDNA3.1-2VNTR/myc-hisB with Lipofectamine2000. The above-mentioned mouse myeloma cells were inoculated subcutaneously into female BALB/c mice for establishing tumor-bearing animal models. These female BALB/c mice were immunized with pcDNA-2VNTR/myc-hisB or pcDNA/myc-hisB. The cytotoxic T lymphocyte (CTL) activity was detected by the LDH method and the spleen lymphocyte proliferation activity was detected by CCK-8 method. RESULTS: After immunization of BALB/c tumor-bearing mice with recombinant plasmid for 25 days, the tumor mass (0.5605 ± 0.2065 g) was significantly lighter than that in the empty plasmid control group (1.521 ± 0.6985 g) (P < 0.01) and the control group (1.5315 ± 0.5425 g) (P < 0.01). The difference of tumor mass was not statislically significant between empty plasmid control group (1.521 ± 0.6985 g) and the control group (1.5315 ± 0.5425 g) (P > 0.05). The CTL and NK cell activity was significantly higher in the group of intramuscular injection with recombinant plasmid than that in control group. The spleen lymphocyte proliferation was statistically significantly increased after being immunized with recombinant plasmid pcDNA3.1-2VNTR/myc-hisB, compared with empty vector (P < 0.01). The results showed that MUC1-2VNTR gene immunization could induce anti-tumor effect in MM tumor-bearing mice. CONCLUSION: MUC1-2VNTR DNA immunization can elicit both humoral and cellular tumor specific immune response to multiple myeloma in MM tumor-bearing mice. It suggested that the MUC1-2VNTR DNA vaccine may be a potential treatment measure for patients with MM.

[Construction of eukaryotic expressing vector of multiple myeloma mucin-1 and its expression in COS-7 cells in vitro].

In order to construct an eukaryotic expression vector for gene of multiple myeloma mucin1 (muc1-2vntr) gene and to express it in COS-7 cells in vitro, so to provide the basic material for further research of multiple myeloma DNA vaccine. muc1-2vntr coding gene was used as a research gene and a KOZAK sequence was inserted before the gene Hind III and XbaI restriction sites were inserted before and after the coding gene. Then the whole sequence was synthesized and inserted into pcDNA3.1/myc-his B vector, and the resulted recombinant vector was transformed into E.coil competent cells to get an engineering strain, the recombinant plasmid pcDNA3.1-2vntr/myc-his B identified by restriction analysis and DNA sequencing were transfected into COS-7 cells by liposome-mediated gene transfer method. Finally, fluorescent microscopy was used to assess GFP expression and Western blot analysis using muc1 monoclonal antibody was used to recognize vntr, confirming the expression of vntr. The results showed that the full length of synthesized muc1-2vntr gene, as expected, was 140 bp. Both restriction analysis and DNA sequencing demonstrated that pcDNA3.1-2vntr/myc-his B included the whole translation frame region and muc1-2vntr gene. Furthermore, the fluorescence microscopy proved that the recombinant plasmid had been successfully transfected into COS-7 cells. The expression of mucin-1 protein was observed both in the transfected cell and the cell supernatant by Western blot. It is concluded that the pcDNA3.1-2vntr/myc-his B has been successfully constructed and expressed in COS-7 cells in vitro, which provides the basic material for further researches of mucin-1 function and possible multiple myloma DNA vaccine.