Particle-based simulations reveal two positive feedback loops allow relocation and stabilization of the polarity site during yeast mating.

Many cells adjust the direction of polarized growth or migration in response to external directional cues. The yeast Saccharomyces cerevisiae orient their cell fronts (also called polarity sites) up pheromone gradients in the course of mating. However, the initial polarity site is often not oriented towards the eventual mating partner, and cells relocate the polarity site in an indecisive manner before developing a stable orientation. During this reorientation phase, the polarity site displays erratic assembly-disassembly behavior and moves around the cell cortex. The mechanisms underlying this dynamic behavior remain poorly understood. Particle-based simulations of the core polarity circuit revealed that molecular-level fluctuations are unlikely to overcome the strong positive feedback required for polarization and generate relocating polarity sites. Surprisingly, inclusion of a second pathway that promotes polarity site orientation generated relocating polarity sites with properties similar to those observed experimentally. This pathway forms a second positive feedback loop involving the recruitment of receptors to the cell membrane and couples polarity establishment to gradient sensing. This second positive feedback loop also allows cells to stabilize their polarity site once the site is aligned with the pheromone gradient.

Thermal tolerance of dried shoots of the moss Bryum argenteum.

We conducted laboratory experiments to determine the lethal temperatures of the shoots of dried Bryum argenteum and to determine how this restoration species responds to extreme environments. We specifically assessed changes in gene expression levels in the shoots of dried B. argenteum plants that were subjected to sudden heat shock (control (20 ± 2°C), 80°C, 100°C, 110°C or 120°C) followed by exposure to heat for an additional 10, 20, 30 or 60 min. After they were exposed to heat, the samples were placed in wet sand medium, and their survival and regeneration abilities were evaluated daily for 56 days. The results showed that lethal temperatures significantly reduced the shoot regeneration potential, delayed both shoot and protonemal emergence times and reduced the protonemal emergence area. In addition, the expression of nine genes (HSF3, HSP70, ERF, LEA, ELIP, LHCA, LHCB, Tr288 and DHN) was induced by temperature stress, as assessed after 30 min of exposure. Additionally, a new thermal tolerance level for dried B. argenteum - 120°C for 20 min - was determined, which was the highest temperature recorded for this moss; this tolerance exceeded the previous record of 110°C for 10 min. These findings help elucidate the survival mechanism of this species under heat shock stress and facilitate the recovery and restoration of destroyed ecosystems.

Differentiation in fructification percentage between two morphs of Amomum tsaoko (Zingiberaceae).

Amomum tsaoko is a flexistylous ginger. Flexistyly is a unique floral mechanism promoting outcrossing, which is known only in some species of Zingiberaceae till date. This is a pioneer report on flexistyly in A. tsaoko from the aspect of fructification percentage to clarify its influence on reproduction. We observed in 2007 and 2008 that the fructification percentage of the anaflexistyled and the cataflexistyled inflorescence were 14.89 ± 10.35% and 11.31 ± 7.91% respectively, with significant difference (d.f. = 141.920, t = 2.518, P = 0.013 < 0.05). The greatly significant difference between 2007 and 2008 were present in both the flower number (d.f. = 93, t = -2.819, P = 0.006 < 0.01) and the fructification percentage (d.f. = 93, t = -2.894, P = 0.005 < 0.01) of the cataflexistylous inflorescence. Although the two morphs were similar in morphological characteristics, there was some gender differentiation between them, showing a possibility that the anaflexistylous morph might function more as females and the cataflexistylous morph more as males. Reproduction of the cataflexistylous morph was significantly sensitive to change of environmental factors, in contrast to the anaflexistylous morph, thus the yield varied between the abundant year (2008) and the off year (2007).

Host shoot clipping depresses the growth of weedy hemiparasitic Pedicularis kansuensis.

Root hemiparasitic plants show optimal growth when attached to a suitable host by abstracting water and nutrients. Despite the fact that damage to host plants in the wild occurs frequently in various forms (e.g. grazing), effects of host damage on growth and physiological performance of root hemiparasites remain unclear. In this study, host shoot clipping was conducted to determine the influence of host damage on photosynthetic and growth performance of a weedy root hemiparasite, Pedicularis kansuensis, and its interaction with a host, Elymus nutans. Photosynthetic capacity, tissue mineral nutrient content and plant biomass of P. kansuensis were significantly improved when attached to a host plant. Host clipping had no effect on quantum efficiency (ΦPSII), but significantly reduced the growth rate and biomass of P. kansuensis. In contrast, clipping significantly improved photosynthetic capacity and accumulation of potassium in E. nutans. No significant decrease in biomass was observed in clipped host plants. By changing nutrient absorption and allocation, clipping affected the interaction between P. kansuensis and its host. Our results showed that host clipping significantly suppressed the growth of weedy P. kansuensis, but did not affect biomass accumulation in E. nutans. We propose that grazing (a dominant way of causing host damage in the field) may have a potential in the control against the weedy hemiparasite.

The responses of the quantitative characteristics of a ramet population of the ephemeroid rhizomatous sedge Carex physodes to the moisture content of the soil in various locations on sand dunes.

In this study, the soil moisture content was measured, and the quantitative characteristics of this sedge species were compared. The phenotypic plasticity of each parameter and the linear regression relationships were analyzed. The results showed that the soil moisture content was significantly affected by location, soil depth, and sampling date. The aboveground biomass, underground biomass, biomass density, and population density at the peak were significantly higher than elsewhere on the dune. However, the morphological plasticity index of the quantitative characteristics was higher at the base and middle of the dune. When the soil moisture content decreased, the underground biomass and ramet biomass density increased. The aboveground and underground biomasses were strongly negatively correlated, but the ramet height and aboveground biomass were strongly positively correlated. These results indicated that the soil water content significantly affected the clonal growth of C. physodes. The responsiveness of C. physodes may be adaptive when the soil resource supply is low. The strong morphological plasticity of the species appears to be ecologically important for the maintenance and dominance of this species in the dune habitat.

Nutrient requirements differ in two Pedicularis species in the absence of a host plant: implication for driving forces in the evolution of host preference of root hemiparasitic plants.

BACKGROUND AND AIMS: Facultative root hemiparasitic plants generally have a wide host range, but in most cases show an obvious host preference. The reasons for the marked difference in growth performance of hemiparasites when attached to different hosts are not fully understood. In this study, the hypothesis was tested that hemiparasites showing a preference for different hosts have different nutrient requirements. METHODS: Two facultative root hemiparasitic Pedicularis species (P. rex and P. tricolor) with a different host dependency and preference were used to test their responses to inorganic solutes. The effects of nitrogen, phosphorus and potassium on growth of the hemiparasitic plants not attached to a host were determined, using an orthogonal design in pot cultivation under greenhouse conditions. Variables including biomass, shoot nutrient concentration, root:shoot (R:S) ratios and the number of haustoria were measured. KEY RESULTS: As in autotrophic plants, nutrient deficiency reduced dry weight (DW) and nutrient concentrations in the root hemiparasites. Nitrogen and phosphorus significantly influenced growth of both Pedicularis species, while potassium availability influenced only shoot DW of P. rex. Nitrogen had far more effect on growth of P. rex than on P. tricolor, while phosphorus deficiency caused more marked growth depression in P. tricolor than in P. rex. Pedicularis rex grew faster than P. tricolor in a range of nutrient supplies. Different patterns of biomass allocation between the two Pedicularis species were observed. While P. rex invested more into roots (particularly fine rootlets) than P. tricolor, the number of haustoria produced by P. rex was relatively much lower than that produced by P. tricolor, which had a much smaller root system. CONCLUSIONS: The two Pedicularis species differ in nutrient requirements and biomass allocation. Distinct interspecific traits in growth and nutrient requirements can be driving forces for the differential interactions between hemiparasites and their hosts.

Direct and indirect influences of arbuscular mycorrhizal fungi on phosphorus uptake by two root hemiparasitic Pedicularis species: do the fungal partners matter at low colonization levels?

BACKGROUND AND AIMS: Because most parasitic plants do not form mycorrhizal associations, the nutritional roles of arbuscular mycorrhizal (AM) fungi in them have hardly been tested. Some facultative root hemiparasitic Pedicularis species form AM associations and hence are ideal for testing both direct and indirect effects of AM fungi on their nutrient acquisition. The aim of this study was to test the influence of AM inoculation on phosphorus (P) uptake by Pedicularis rex and P. tricolor. METHODS: (32)P labelling was used in compartmented pots to assess the contribution of the AM pathway and the influence of AM inoculation on P uptake from a host plant into the root hemiparasites. Laboratory isolates of fungal species (Glomus mosseae and G. intraradices) and the host species (Hordeum vulgare 'Fleet') to which the two Pedicularis species showed obvious responses in haustorium formation and growth in previous studies were used. KEY RESULTS: The AM colonization of both Pedicularis spp. was low (<15 % root length) and only a very small proportion of total plant P (<1 %) was delivered from the soil via the AM fungus. In a separate experiment, inoculation with AM fungi strongly interfered with P acquisition by both Pedicularis species from their host barley, almost certainly because the numbers of haustoria formed by the parasite were significantly reduced in AM plants. CONCLUSIONS: Roles of AM fungi in nutrient acquisition by root parasitic plants were quantitatively demonstrated for the first time. Evidence was obtained for a novel mechanism of preventing root parasitic plants from overexploiting host resources through AM fungal-induced suppression of the absorptive structures in the parasites.

Flowering phenology of tree rhododendron along an elevation gradient in two sites in the Eastern Himalayas.

Flowering phenology of tree rhododendron (Rhododendron arboreum Sm.) was monitored in situ along elevation gradients in two distinct ecological settings. Observations were carried out in Gaoligong Nature Reserve (GNR) in China and in the Kanchenjunga Conservation Area (KCA) in Nepal. Using the crown density method, flowering events of the selected species were recorded. Flowering duration and synchrony were determined within each site and along the elevation gradient in each study area. Our observations showed high synchrony throughout the elevation gradient, especially for peak flowering. Mean 15-day soil temperature, soil parameters (soil moisture, nitrogen, organic matter and pH), age of the observed trees, and site characteristics (litter cover, canopy cover, inclination) were related to mean initial and peak flowering dates using partial least squares regression (PLS). Results differed between the two sites, but winter temperature was the most important variable affecting the regression model for both initial flowering and peak flowering at both sites. After temperature, soil moisture was the most important variable for explaining initial flowering dates. The distribution of tree rhododendron indicates that it is able to grow in a wide range of habitats with different environmental conditions. The recent trend of rising winter-spring temperature and the detected bloom-advancing effect of high temperatures during this period suggest that tree rhododendron might expand its distributional range in response to global warming.

Inoculation with arbuscular mycorrhizal fungi suppresses initiation of haustoria in the root hemiparasite Pedicularis tricolor.

BACKGROUND AND AIMS: Plant parasitism and arbuscular mycorrhizal (AM) associations have many parallels and share a number of regulatory pathways. Despite a rapid increase in investigations addressing the roles of AM fungi in regulating interactions between parasitic plants and their hosts, few studies have tested the effect of AM fungi on the initiation and differentiation of haustoria, the parasite-specific structures exclusively responsible for host attachment and nutrient transfer. In this study, we tested the influence of AM fungi on haustorium formation in a root hemiparasitic plant. METHODS: Using a facultative root hemiparasitic species (Pedicularis tricolor) with the potential to form AM associations, the effects of inoculation were tested with two AM fungal species, Glomus mosseae and Glomus intraradices, on haustorium initiation in P. tricolor grown alone or with Hordeum vulgare 'Fleet' (barley) as the host plant. This study consisted of two greenhouse pot experiments. KEY RESULTS: Both AM fungal species dramatically suppressed intraspecific haustorium initiation in P. tricolor at a very low colonization level. The suppression over-rode inductive effects of the parasite's host plant on haustoria production and caused significant growth depression of P. tricolor. CONCLUSIONS: AM fungi had strong and direct suppressive effects on haustorium formation in the root hemiparasite. The significant role of AM fungi in haustorium initiation of parasitic plants was demonstrated for the first time. This study provides new clues for the regulation of haustorium formation and a route to development of new biocontrol strategies in management of parasitic weeds.

A Legume Host Benefits More from Arbuscular Mycorrhizal Fungi Than a Grass Host in the Presence of a Root Hemiparasitic Plant.

In nature, most plants parasitized by root hemiparasites are also colonized by mutualistic arbuscular mycorrhizal (AM) fungi, highlighting the prevalence of this tripartite interaction. AM colonization is generally found to improve the growth of parasitized legumes but has little impact on grass hosts parasitized by root hemiparasites, and the underlying mechanisms are still unclear. In this study, we conducted a pot experiment to test the influence of AM fungus (Glomus mosseae) on the growth and photosynthesis of leguminous Trifolium repens and gramineous Elymus nutans in the presence of a root hemiparasitic plant (Pedicularis kansuensis). The results showed that inoculation with AM fungi significantly improved the growth performance of parasitized legumes via enhancing their nutrient status and photosynthetic capacity, even though a larger P. kansuensis parasitized the legume host in the AM treatment. In contrast, AM colonization slightly improved the shoot DW of grass hosts by suppressing haustoria formation and the growth of P. kansuensis. Our results demonstrated that legume hosts benefit more from AM inoculation than grass hosts in the presence of hemiparasitic plants, and set out the various mechanisms. This study provides new clues for parsing the tritrophic interaction of AM fungi, parasitic plants, and host plants.

Molecular phylogeny of the subgenus Ceratotropis (genus Vigna, Leguminosae) reveals three eco-geographical groups and Late Pliocene-Pleistocene diversification: evidence from four plastid DNA region sequences.

BACKGROUND AND AIMS: The subgenus Ceratotropis in the genus Vigna is widely distributed from the Himalayan highlands to South, Southeast and East Asia. However, the interspecific and geographical relationships of its members are poorly understood. This study investigates the phylogeny and biogeography of the subgenus Ceratotropis using chloroplast DNA sequence data. METHODS: Sequence data from four intergenic spacer regions (petA-psbJ, psbD-trnT, trnT-trnE and trnT-trnL) of chloroplast DNA, alone and in combination, were analysed using Bayesian and parsimony methods. Divergence times for major clades were estimated with penalized likelihood. Character evolution was examined by means of parsimony optimization and MacClade. KEY RESULTS: Parsimony and Bayesian phylogenetic analyses on the combined data demonstrated well-resolved species relationships in which 18 Vigna species were divided into two major geographical clades: the East Asia-Southeast Asian clade and the Indian subcontinent clade. Within these two clades, three well-supported eco-geographical groups, temperate and subtropical (the East Asia-Southeast Asian clade) and tropical (the Indian subcontinent clade), are recognized. The temperate group consists of V. minima, V. nepalensis and V. angularis. The subtropical group comprises the V. nakashimae-V. riukiuensis-V. minima subgroup and the V. hirtella-V. exilis-V. umbellata subgroup. The tropical group contains two subgroups: the V. trinervia-V. reflexo-pilosa-V. trilobata subgroup and the V. mungo-V. grandiflora subgroup. An evolutionary rate analysis estimated the divergence time between the East Asia-Southeast Asia clade and the Indian subcontinent clade as 3·62 ± 0·3 million years, and that between the temperate and subtropical groups as 2·0 ± 0·2 million years. CONCLUSIONS: The findings provide an improved understanding of the interspecific relationships, and ecological and geographical phylogenetic structure of the subgenus Ceratotropis. The quaternary diversification of the subgenus Ceratotropis implicates its geographical dispersal in the south-eastern part of Asia involving adaptation to climatic condition after the collision of the Indian subcontinent with the Asian plate. The phylogenetic results indicate that the epigeal germination is plesiomorphic, and the germination type evolved independently multiple times in this subgenus, implying its limited taxonomic utility.

Leaf anatomical structures of Paphiopedilum and Cypripedium and their adaptive significance.

Paphiopedilum and Cypripedium are closely related in phylogeny, but have contrasting leaf traits and habitats. To understand the divergence in leaf traits of Paphiopedilum and Cypripedium and their adaptive significance, we analyzed the leaf anatomical structures, leaf dry mass per area (LMA), leaf lifespan (LL), leaf nitrogen concentration (N (mass)), leaf phosphorus concentration (P (mass)), mass-based light-saturated photosynthetic rate (A (mass)), water use efficiency (WUE), photosynthetic nitrogen use efficiency (PNUE) and leaf construction cost (CC) for six species. Compared with Cypripedium, Paphiopedilum was characterized by drought tolerance derived from its leaf anatomical structures, including fleshy leaves, thick surface cuticles, huge adaxial epidermis cells, lower total stoma area, and sunken stomata. The special leaf structures of Paphiopedilum were accompanied by longer LL; higher LMA, WUE, and CC; and lower N (mass), P (mass), A (mass), and PNUE compared with Cypripedium. Leaf traits in Paphiopedilum helped it adapt to arid and nutrient-poor karst habitats. However, the leaf traits of Cypripedium reflect adaptations to an environment characterized by rich soil, abundant soil water, and significant seasonal fluctuations in temperature and precipitation. The present results contribute to our understanding of the divergent adaptation of leaf traits in slipper orchids, which is beneficial for the conservation of endangered orchids.

Isolation and characterization of microsatellite loci for Hibiscus aridicola (Malvaceae), an endangered plant endemic to the dry-hot valleys of Jinsha River in Southwest China.

Hibiscus aridicola (Malvaceae) is an endangered ornamental shrub endemic to the dry-hot valleys of Jinsha River in southwest China. Only four natural populations of H. aridicola exist in the wild according to our field investigation. It can be inferred that H. aridicola is facing a very high risk of extinction in the wild and an urgent conservation strategy is required. By using a modified biotin-streptavidin capture method, a total of 40 microsatellite markers were developed and characterized in H. aridicola for the first time. Polymorphisms were evaluated in 39 individuals from four natural populations. Fifteen of the markers showed polymorphisms with two to six alleles per locus; the observed heterozygosity ranged from 0.19 to 0.72. These microsatellite loci would be useful tools for population genetics studies on H. aridicola and other con-generic species which are important to the conservation and development of endangered species.

Hepatic macrophages act as a central hub for relaxin-mediated alleviation of liver fibrosis.

Relaxin is an antifibrotic peptide hormone previously assumed to directly reverse the activation of hepatic stellate cells for liver fibrosis resolution. Using nanoparticle-mediated delivery, here we show that, although relaxin gene therapy reduces liver fibrosis in vivo, in vitro treatment fails to induce quiescence of the activated hepatic stellate cells. We show that hepatic macrophages express the primary relaxin receptor, and that, on relaxin binding, they switch from the profibrogenic to the pro-resolution phenotype. The latter releases exosomes that promote the relaxin-mediated quiescence of activated hepatic stellate cells through miR-30a-5p. Building on these results, we developed lipid nanoparticles that preferentially target activated hepatic stellate cells in the fibrotic liver and encapsulate the relaxin gene and miR-30a-5p mimic. The combinatorial gene therapy achieves synergistic antifibrosis effects in models of mouse liver fibrosis. Collectively, our findings highlight the key role that macrophages play in the relaxin-primed alleviation of liver fibrosis and demonstrate a proof-of-concept approach to devise antifibrotic strategies through the complementary application of nanotechnology and basic science.

Relaxin-FOLFOX-IL-12 triple combination therapy engages memory response and achieves long-term survival in colorectal cancer liver metastasis.

Induction of memory T cell response is inefficient in colorectal cancer (CRC) liver metastasis following existing therapies due to abundant stroma and immunosuppressive environment within the metastatic liver, which leads to fast disease progression, high recurrence rate, and short survival. Two fundamental steps are involved to elicit extensive memory T cell response: stimulation of naive T cells with robust and persistent antigen signals; and maintenance of differentiated memory T cells with survival factors. Here, we demonstrate a rational design of triple combination regimen, including relaxin (RLN), FOLFOX (combination of 5-fluorouracil, leucovorin, and oxaliplatin), and IL-12, successfully stimulates central memory T cells and achieves long-term survival in an aggressive experimental CRC liver metastasis model. Sequential administration of FOLFOX and IL-12 gene therapy following stromal deactivation by RLN gene therapy completely cured established CRC liver metastases in ~50% of mice and provided long-lasting protection against tumor recurrence. The study here may highlight the potential of evoking memory response as a curative therapy for the treatment of CRC liver metastasis.

Nano-puerarin regulates tumor microenvironment and facilitates chemo- and immunotherapy in murine triple negative breast cancer model.

Tumor associated fibroblasts (TAFs) are key stromal cells mediating the desmoplastic reaction and being partially responsible for the drug-resistance and immunosuppressive microenvironment formation in solid tumors. Delivery of genotoxic drugs off-targetedly to kill TAFs results in production of Wnt16 which renders the neighboring tumor cells drug resistant as shown in our previous study (PMC4623876). Our current approach looks for means to deactivate, rather than kill, TAFs. Reactive oxygen species (ROS) are the central hub of multiple profibrogenic pathways and indispensable for TAFs activation. Herein, puerarin was identified to effectively downregulate ROS production in the activated myofibroblast. In this study, a novel puerarin nanoemulsion (nanoPue) was developed to improve the solubility and bioavailability of puerarin. NanoPue significantly deactivated the stromal microenvironment (e.g., ~6-fold reduction of TAFs in nanoPue treated mice compared with the PBS control, p < 0.0001) and facilitated chemotherapy effect of nano-paclitaxel in the desmoplastic triple-negative breast cancer (TNBC) model. Moreover, the removal of the physical barrier increased intra-tumoral infiltration of cytotoxic T cell by 2-fold. This activated immune microenvironment allowed nanoPue to synergize PD-L1 blockade therapy in TNBC model.

ScDREB8, a novel A-5 type of DREB gene in the desert moss Syntrichia caninervis, confers salt tolerance to Arabidopsis.

Salinity is a major limitation factor for crop productivity worldwide. DREB transcription factors broadly participate in plant stress response and have been extensively identified in a wide variety of plants. In this study, we characterized and analyzed the function of a novel A-5 type DREB gene ScDREB8 from the desiccation tolerant moss Syntrichia caninervis. Yeast one-hybrid experiment showed that ScDREB8 had no transactivation activity. Transient expression assay in onion epidermal cells revealed that ScDREB8 is distributed throughout the cell with no apparent specificity. Overexpression of ScDREB8 significantly increased the germination rate of Arabidopsis under salt stress and improved the salt tolerance of Arabidopsis at the seedling stage by up-regulating the expression of downstream stress-related genes and improving ROS scavenging ability. ScDREB8 is a promising candidate gene for improving crop salt stress and will provide greater insight to the molecular mechanism of stress tolerance of A-5 type DREB proteins.

N-P Fertilization Inhibits Growth of Root Hemiparasite Pedicularis kansuensis in Natural Grassland.

Fertilization has been shown to affect interactions between root hemiparasitic plants and their host plants, alleviating damage to the hosts by parasitism. However, as a majority of studies were conducted in pot cultivation, the influence of fertilizer application on root hemiparasites and the surrounding plant community in field conditions as well as relevant mechanisms remain unclear. We manipulated soil nutrient resources in a semi-arid subalpine grassland in the Tianshan Mountains, northwestern China, to explore the links between fertilization and plant community composition, productivity, survival, and growth of a weedy root hemiparasite (Pedicularis kansuensis). Nitrogen (at a low rate, LN, 30 kg N ha-1 year-1 as urea; or at a high rate, HN, 90 kg N ha-1 year-1 as urea) and phosphorus [100 kg ha-1 year-1 as Ca(H2PO4)2⋅H2O] were added during two growing seasons. Patterns of foliar nutrient balances were described with isometric log ratios for the different plant functional groups receiving these fertilization regimes. Fertilization with LN, HN, and P reduced above-ground biomass of P. kansuensis, with above-ground biomass in the fertilization treatments, respectively, 12, 1, and 39% of the value found in the unfertilized control. Up to three times more above-ground biomass was produced in graminoids receiving fertilizers, whereas forb above-ground biomass was virtually unchanged by the fertilization regimes and forb species richness was reduced by 52% in the HN treatment. Fertilization altered foliar nutrient balances, and distinct patterns emerged for each plant functional group. Foliar [C | P,N] balance in the plant community was negatively correlated with above-ground biomass (P = 0.03). The inhibited competitiveness of P. kansuensis, which showed a much higher [C | P,N] balance, could be attributed to reduced C assimilation rather than mineral nutrient acquisition, as shown by significant increase in foliar N and P concentrations but little increase in C concentration following fertilization.

Phylogenetic reassessment of tribe Anemoneae (Ranunculaceae): Non-monophyly of Anemone s.l. revealed by plastid datasets.

Morphological and molecular evidence strongly supported the monophyly of tribe Anemoneae DC.; however, phylogenetic relationships among genera of this tribe have still not been fully resolved. In this study, we sampled 120 specimens representing 82 taxa of tribe Anemoneae. One nuclear ribosomal internal transcribed spacer (nrITS) and six plastid markers (atpB-rbcL, matK, psbA-trnQ, rpoB-trnC, rbcL and rps16) were amplified and sequenced. Both Maximum likelihood and Bayesian inference methods were used to reconstruct phylogenies for this tribe. Individual datasets supported all traditional genera as monophyletic, except Anemone and Clematis that were polyphyletic and paraphyletic, respectively, and revealed that the seven single-gene datasets can be split into two groups, i.e. nrITS + atpB-rbcL and the remaining five plastid markers. The combined nrITS + atpB-rbcL dataset recovered monophyly of subtribes Anemoninae (i.e. Anemone s.l.) and Clematidinae (including Anemoclema), respectively. However, the concatenated plastid dataset showed that one group of subtribes Anemoninae (Hepatica and Anemone spp. from subgenus Anemonidium) close to the clade Clematis s.l. + Anemoclema. Our results strongly supported a close relationship between Anemoclema and Clematis s.l., which included Archiclematis and Naravelia. Non-monophyly of Anemone s.l. using the plastid dataset indicates to revise as two genera, new Anemone s.l. (including Pulsatilla, Barneoudia, Oreithales and Knowltonia), Hepatica (corresponding to Anemone subgenus Anemonidium).

Novel DREB A-5 subgroup transcription factors from desert moss (Syntrichia caninervis) confers multiple abiotic stress tolerance to yeast.

Syntrichia caninervis Mitt. is a typical desiccation tolerant moss from a temperate desert which has been a good resource for stress tolerant gene isolation. Dehydration responsive element binding proteins (DREBs) was proven to play an important role in responding to abiotic stress, which has been identified in many plants, and were rarely reported in moss. In this study, we cloned ten DREB genes from S. caninervis, and investigated their abiotic stress response and stress tolerance. The results showed that ten ScDREB proteins belonged to the A-5 sub-group of the DREB sub-family. Six genes, ScDREB1, ScDREB2, ScDREB4, ScDREB6, ScDREB7, and ScDREB8 were involved in the ABA-dependent signal pathway and the desiccation, salt, and cold stress response. ScDREB3 also responded to desiccation, salt, and cold stresses, but was insensitive to ABA treatment. Another gene, ScDREB5, was involved in an ABA-independent cold stress-responsive signal pathway. Two genes, ScDREB9 and ScDREB10, responded slightly or had no response to neither stress factor or ABA treatment. We transformed four typical genes into yeast cells and the stress tolerance ability of transgenic yeast was evaluated. The results showed that ScDREB3 and ScDREB5 enhanced the yeast's cold and salt tolerance. ScDREB8 and ScDREB10 conferred the osmotic, salt, cold, and high temperature stresses tolerance, especially for osmotic and salt stresses. Our results indicated that A-5 sub-group DREB genes in S. caninervis played important roles in abiotic stresses response and enhanced stress tolerance to transgenic yeast. To our knowledge, this is the first report on DREB genes characterization from desiccation tolerant moss, and this study will not only provide insight into the molecular mechanisms of S. caninervis adaptation to environmental stresses, but also provides valuable gene candidates for plant molecular breeding.