Appropriate Reference Genes for RT-qPCR Normalization in Various Organs of Anemone flaccida Fr. Schmidt at Different Growing Stages.

Anemone flaccida Fr. Schmidt is a traditional medicinal herb in southwestern China and has multiple pharmacological effects on bruise injuries and rheumatoid arthritis (RA). A new drug with a good curative effect on RA has recently been developed from the extract of A. flaccida rhizomes, of which the main medicinal ingredients are triterpenoid saponins. Due to excessive exploitation, the wild population has been scarce and endangered in a few of its natural habitats and research on the cultivation of the plant commenced. Studies on the gene expressions related to the biosynthesis of triterpenoid saponins are not only helpful for understanding the effects of environmental factors on the medicinal ingredient accumulations but also necessary for monitoring the herb quality of the cultivated plants. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a sensitive and powerful technique has been widely used to detect gene expression across tissues in plants at different stages; however, its accuracy and reliability depend largely on the reference gene selection. In this study, the expressions of 10 candidate reference genes were evaluated in various organs of the wild and cultivated plants at different stages, using the algorithms of geNorm, NormFinder and BestKeeper, respectively. The purpose of this study was to identify the suitable reference genes for RT-qPCR detection in A. flaccida. The results showed that two reference genes were sufficient for RT-qPCR data normalization in A. flaccida. PUBQ and ETIF1a can be used as suitable reference genes in most organs at various stages because of their expression stabilitywhereas the PUBQ and EF1Α genes were desirable in the rhizomes of the plant at the vegetative stage.

The Double-Layer Method to the Genesis of Androgenic Plants in Anemone coronaria.

Homozygous lines occur for plant breeding programs and for studies about gene expression and genetic mapping and they can be derived from anther culture. In this chapter, the method to obtain androgenic plants from an ornamental cut flower, Anemone coronaria belonging to the Ranunculaceae family, is described. In this species, androgenic plants were obtained culturing anthers with responsive microspores in Petri dishes containing a double layer of substrate with specific composition. Moreover, thermic treatment has been applied to induce the switch from pollen development program to embryo development program. The method allows to produce both double-haploid plants from diploid mothers (2n) and di-haploid plants from tetraploid mothers (4n).

Spiral phyllotaxis underlies constrained variation in Anemone (Ranunculaceae) tepal arrangement.

Stabilization and variation of floral structures are indispensable for plant reproduction and evolution; however, the developmental mechanism regulating their structural robustness is largely unknown. To investigate this mechanism, we examined positional arrangement (aestivation) of excessively produced perianth organs (tepals) of six- and seven-tepaled (lobed) flowers in six Anemone species (Ranunculaceae). We found that the tepal arrangement that occurred in nature varied intraspecifically between spiral and whorled arrangements. Moreover, among the studied species, variation was commonly limited to three types, including whorls, despite five geometrically possible arrangements in six-tepaled flowers and two types among six possibilities in seven-tepaled flowers. A spiral arrangement, on the other hand, was unique to five-tepaled flowers. A spiral phyllotaxis model with stochasticity on initiating excessive primordia accounted for these limited variations in arrangement in cases when the divergence angle between preexisting primordia was less than 144°. Moreover, interspecific differences in the frequency of the observed arrangements were explained by the change of model parameters that represent meristematic growth and differential organ growth. These findings suggest that the phyllotaxis parameters are responsible for not only intraspecific stability but interspecific difference of floral structure. Decreasing arrangements from six-tepaled to seven-tepaled Anemone flowers demonstrate that the stabilization occurs as development proceeds to increase the component (organ) number, in contrast from the intuition that the variation will be larger due to increasing number of possible states (arrangements).

Biotic and abiotic drivers of intraspecific trait variation within plant populations of three herbaceous plant species along a latitudinal gradient.

BACKGROUND: The importance of intraspecific trait variation (ITV) is increasingly acknowledged among plant ecologists. However, our understanding of what drives ITV between individual plants (ITVBI) at the population level is still limited. Contrasting theoretical hypotheses state that ITVBI can be either suppressed (stress-reduced plasticity hypothesis) or enhanced (stress-induced variability hypothesis) under high abiotic stress. Similarly, other hypotheses predict either suppressed (niche packing hypothesis) or enhanced ITVBI (individual variation hypothesis) under high niche packing in species rich communities. In this study we assess the relative effects of both abiotic and biotic niche effects on ITVBI of four functional traits (leaf area, specific leaf area, plant height and seed mass), for three herbaceous plant species across a 2300 km long gradient in Europe. The study species were the slow colonizing Anemone nemorosa, a species with intermediate colonization rates, Milium effusum, and the fast colonizing, non-native Impatiens glandulifera. RESULTS: Climatic stress consistently increased ITVBI across species and traits. Soil nutrient stress, on the other hand, reduced ITVBI for A. nemorosa and I. glandulifera, but had a reversed effect for M. effusum. We furthermore observed a reversed effect of high niche packing on ITVBI for the fast colonizing non-native I. glandulifera (increased ITVBI), as compared to the slow colonizing native A. nemorosa and M. effusum (reduced ITVBI). Additionally, ITVBI in the fast colonizing species tended to be highest for the vegetative traits plant height and leaf area, but lowest for the measured generative trait seed mass. CONCLUSIONS: This study shows that stress can both reduce and increase ITVBI, seemingly supporting both the stress-reduced plasticity and stress-induced variability hypotheses. Similarly, niche packing effects on ITVBI supported both the niche packing hypothesis and the individual variation hypothesis. These results clearly illustrates the importance of simultaneously evaluating both abiotic and biotic factors on ITVBI. This study adds to the growing realization that within-population trait variation should not be ignored and can provide valuable ecological insights.

Natural selection and neutral evolution jointly drive population divergence between alpine and lowland ecotypes of the allopolyploid plant Anemone multifida (Ranunculaceae).

Population differentiation can be driven in large part by natural selection, but selectively neutral evolution can play a prominent role in shaping patters of population divergence. The decomposition of the evolutionary history of populations into the relative effects of natural selection and selectively neutral evolution enables an understanding of the causes of population divergence and adaptation. In this study, we examined heterogeneous genomic divergence between alpine and lowland ecotypes of the allopolyploid plant, Anemone multifida. Using peak height and dominant AFLP data, we quantified population differentiation at non-outlier (neutral) and outlier loci to determine the potential contribution of natural selection and selectively neutral evolution to population divergence. We found 13 candidate loci, corresponding to 2.7% of loci, with signatures of divergent natural selection between alpine and lowland populations and between alpine populations (Fst  = 0.074-0.445 at outlier loci), but neutral population differentiation was also evident between alpine populations (FST  = 0.041-0.095 at neutral loci). By examining population structure at both neutral and outlier loci, we determined that the combined effects of selection and neutral evolution are associated with the divergence of alpine populations, which may be linked to extreme abiotic conditions and isolation between alpine sites. The presence of outlier levels of genetic variation in structured populations underscores the importance of separately analyzing neutral and outlier loci to infer the relative role of divergent natural selection and neutral evolution in population divergence.

Tree litter and forest understorey vegetation: a conceptual framework to understand the effects of tree litter on a perennial geophyte, Anemone nemorosa.

BACKGROUND AND AIMS: Litter is a key factor in structuring plant populations, through positive or negative interactions. The litter layer forms a mechanical barrier that is often strongly selective against individuals lacking hypocotyle plasticity. Litter composition also interacts with plant growth by providing beneficial nutrients or, inversely, by allowing harmful allelopathic leaching. As conspicuous litter fall accumulation is often observed under deciduous forests, interactions between tree litter and understorey plant populations are worthy of study. METHODS: In a 1-year ex-situ experiment, the effects of tree litter on the growth of Anemone nemorosa, a small perennial forest geophyte, were investigated. Three 'litter quantity' treatments were defined, representative of forest floor litter (199, 356·5 and 514 g m(-2)), which were crossed with five 'litter composition' treatments (Quercus petraea, Fagus sylvatica, Carpinus betulus, Q. petraea + F. sylvatica and Q. petraea + C. betulus), plus a no-litter control. Path analysis was then used to investigate the pathways linking litter characteristics and components of adult plant growth. KEY RESULTS: As expected, the heavier the litter, the longer the petiole; rhizome growth, however, was not depreciated by the litter-induced petiole lengthening. Both rhizome mass increment and number of initiated buds marginally increased with the amount of litter. Rhizome mass increment was in fact determined primarily by leaf area and leaf life span, neither of which was unequivocally correlated with any litter characteristics. However, the presence of litter significantly increased leafing success: following a late frost event, control rhizomes growing in the absence of litter experienced higher leaf mortality before leaf unfolding. CONCLUSIONS: The study questions the role of litter as a physical or chemical barrier to ground vegetation; to better understand this role, there is a need for ex-situ, longer-term experiments coupled with in-situ observations in the forest.

The response of forest plant regeneration to temperature variation along a latitudinal gradient.

BACKGROUND AND AIMS: The response of forest herb regeneration from seed to temperature variations across latitudes was experimentally assessed in order to forecast the likely response of understorey community dynamics to climate warming. METHODS: Seeds of two characteristic forest plants (Anemone nemorosa and Milium effusum) were collected in natural populations along a latitudinal gradient from northern France to northern Sweden and exposed to three temperature regimes in growth chambers (first experiment). To test the importance of local adaptation, reciprocal transplants were also made of adult individuals that originated from the same populations in three common gardens located in southern, central and northern sites along the same gradient, and the resulting seeds were germinated (second experiment). Seedling establishment was quantified by measuring the timing and percentage of seedling emergence, and seedling biomass in both experiments. KEY RESULTS: Spring warming increased emergence rates and seedling growth in the early-flowering forb A. nemorosa. Seedlings of the summer-flowering grass M. effusum originating from northern populations responded more strongly in terms of biomass growth to temperature than southern populations. The above-ground biomass of the seedlings of both species decreased with increasing latitude of origin, irrespective of whether seeds were collected from natural populations or from the common gardens. The emergence percentage decreased with increasing home-away distance in seeds from the transplant experiment, suggesting that the maternal plants were locally adapted. CONCLUSIONS: Decreasing seedling emergence and growth were found from the centre to the northern edge of the distribution range for both species. Stronger responses to temperature variation in seedling growth of the grass M. effusum in the north may offer a way to cope with environmental change. The results further suggest that climate warming might differentially affect seedling establishment of understorey plants across their distribution range and thus alter future understorey plant dynamics.

For she that hath, to her shall be given Implications of flowering in Anemone nemorosa L.

We looked for life-history trade-offs between flowering, vegetative growth and somatic maintenance in the common woodland herb Anemone nemorosa. A. nemorosa forms a horizontal rhizome system consisting of previously formed annual segments and terminated by a flowering or non-flowering shoot. Resources acquired by the aboveground parts are used for flowering, seed production, storage and growth of the annual segments. Resources stored in the rhizome during the growing period are used for preformation of buds, somatic maintenance between two growing periods and development of aboveground parts in the following spring. We hypothesised that the decision to invest in flower buds depends on the amount of resources stored in the recently formed annual segment. We also hypothesised a trade-off between flowering and segment growth and, finally, as a consequence, we expected individual rhizomes to alternate between the flowering and the non-flowering state. We found that segments producing flower buds were significantly longer than non-flowering segments, indicating that resource level influences the function of the preformed buds. Contrary to our expectations, we found flowering rhizomes produced longer annual segments than non-flowering rhizomes. We suggest the larger leaf area of flowering rhizomes and occasional abortion of flowers or seeds as possible mechanisms behind this pattern. Our study shows that even though the decision to produce a flower bud is taken in another time-frame than that in which the actual flowering and fruiting takes place, an ostensibly inexpedient decision is changed to a neutral or even an advantageous incident.

Plasticity in response to phosphorus and light availability in four forest herbs.

The differential ability of forest herbs to colonize secondary forests on former agricultural land is generally attributed to different rates of dispersal. After propagule arrival, however, establishing individuals still have to cope with abiotic soil legacies from former agricultural land use. We focused on the plastic responses of forest herbs to increased phosphorus availability, as phosphorus is commonly found to be persistently bioavailable in post-agricultural forest soils. In a pot experiment performed under field conditions, we applied three P levels to four forest herbs with contrasting colonization capacities: Anemone nemorosa, Primula elatior, Circaea lutetiana and Geum urbanum. To test interactions with light availability, half of the replicas were covered with shade cloths. After two growing seasons, we measured aboveground P uptake as well as vegetative and regenerative performance. We hypothesized that fast-colonizing species respond the most opportunistically to increased P availability, and that a low light availability can mask the effects of P on performance. All species showed a significant increase in P uptake in the aboveground biomass. The addition of P had a positive effect on the vegetative performances of two of the species, although this was unrelated to their colonization capacities. The regenerative performance was affected by light availability (not by P addition) and was related to the species' phenology. Forest herbs can obviously benefit from the increased availability of P in post-agricultural forests, but not all species respond in the same way. Such differential patterns of plasticity may be important in community dynamics, as they affect the interactions among species.

Elevated CO2 reduces losses of plant diversity caused by nitrogen deposition.

The interactive effects of rising atmospheric carbon dioxide (CO2) concentrations and elevated nitrogen (N) deposition on plant diversity are not well understood. This is of concern because both factors are important components of global environmental change and because each might suppress diversity, with their combined effects possibly additive or synergistic. In a long-term open-air experiment, grassland assemblages planted with 16 species were grown under all combinations of ambient and elevated CO2 and ambient and elevated N. Over 10 years, elevated N reduced species richness by 16% at ambient CO2 but by just 8% at elevated CO2. This resulted from multiple effects of CO2 and N on plant traits and soil resources that altered competitive interactions among species. Elevated CO2 thus ameliorated the negative effects of N enrichment on species richness.

Community-wide ramifications of an associational refuge on shallow rocky reefs.

Little attention has been given to associational refuges in ecology, despite their potential for maintaining species diversity and supporting higher trophic levels. Here I show how the colonial anemone, Corynactis californica, creates a refuge for benthic macroalgae and invertebrate fish prey on intensively grazed shallow rocky reefs in the Santa Barbara Channel off southern California, U.S.A. On reefs heavily grazed by sea urchins, benthic macroalgae and invertebrate fish prey were relatively more abundant among Corynactis colonies than adjacent areas lacking the anemone. Results from field experiments showed that Corynactis facilitated the recruitment of macroalgae and tubicolous amphipods in "urchin-barren" areas subjected to intensive grazing. In areas forested by giant kelp (Macrocystis pyrifera), where grazing intensity from urchins was low, Corynactis suppressed algal recruitment but facilitated tubicolous amphipods. A manipulation of fish and sea urchins suggested that grazing by urchins, as opposed to predation from fish (primarily surfperch Embiotocidae), suppressed tubicolous amphipods, and this activity was hindered by the presence of Corynactis. In systems where human activity has intensified herbivory, associational refuges may maintain species diversity and support higher trophic levels.

Inhibition of the gravitropic bending response of flowering shoots by salicylic acid.

The upward gravitropic bending of cut snapdragon, lupinus and anemone flowering shoots was inhibited by salicylic acid (SA) applied at 0.5 mM and above. This effect was probably not due to acidification of the cytoplasm, since other weak acids did not inhibit bending of snapdragon shoots. In order to study its mode of inhibitory action, we have examined in cut snapdragon shoots the effect of SA on three processes of the gravity-signaling pathway, including: amyloplast sedimentation, formation of ethylene gradient across the stem, and differential growth response. The results show that 1 mM SA inhibited differential ethylene production rates across the horizontal stem and the gravity-induced growth, without significantly inhibiting vertical growth or amyloplast sedimentation following horizontal placement. However, 5 mM SA inhibited all three gravity-induced processes, as well as the growth of vertical shoots, while increasing flower wilting. It may, therefore, be concluded that SA inhibits bending of various cut flowering shoots in a concentration-dependent manner. Thus, at a low concentration SA exerts its effect in snapdragon shoots by inhibiting processes operating downstream to stimulus sensing exerted by amyloplast sedimentation. At a higher concentration SA inhibits bending probably by exerting general negative effects on various cellular processes.

The timing of life-history events in a changing climate.

Although empirical and theoretical studies suggest that climate influences the timing of life-history events in animals and plants, correlations between climate and the timing of events such as egg-laying, migration or flowering do not reveal the mechanisms by which natural selection operates on life-history events. We present a general autoregressive model of the timing of life-history events in relation to variation in global climate that, like autoregressive models of population dynamics, allows for a more mechanistic understanding of the roles of climate, resources and competition. We applied the model to data on 50 years of annual dates of first flowering by three species of plants in 26 populations covering 4 degrees of latitude in Norway. In agreement with earlier studies, plants in most populations and all three species bloomed earlier following warmer winters. Moreover, our model revealed that earlier blooming reflected increasing influences of resources and density-dependent population limitation under climatic warming. The insights available from the application of this model to phenological data in other taxa will contribute to our understanding of the roles of endogenous versus exogenous processes in the evolution of the timing of life-history events in a changing climate.