Pollinator availability as a determinant of flowering time in ocotillo (Fouquieria splendens)

Summary Ocotillo, a perennial shrub of Sonoran and Chihuahuan Deserts, produces its red tubular flowers in spring. This timing coincides with northward migration of hummingbirds through desert areas. Observations of visitors, pollen collections, and seed set reductions following exclusion of different flower visitors indicate that both hummingbirds and solitary bees pollinate ocotillo in southern Arizona. Seed sets of flowers on marked plants varied considerably within and between years, and this variation was related to the temporal match between flowering and hummingbird migration. This suggests that selection acts on plants to synchronize flowering with periods of pollinator abundance.     

New frontiers in competition for pollination

Background

Co-flowering plant species frequently share pollinators. Pollinator sharing is often detrimental to one or more of these species, leading to competition for pollination. Perhaps because it offers an intriguing juxtaposition of ecological opposites – mutualism and competition – within one relatively tractable system, competition for pollination has captured the interest of ecologists for over a century.

Scope

Our intent is to contemplate exciting areas for further work on competition for pollination, rather than to exhaustively review past studies. After a brief historical summary, we present a conceptual framework that incorporates many aspects of competition for pollination, involving both the quantity and quality of pollination services, and both female and male sex functions of flowers. Using this framework, we contemplate a relatively subtle mechanism of competition involving pollen loss, and consider how competition might affect plant mating systems, overall reproductive success and multi-species interactions. We next consider how competition for pollination might be altered by several emerging consequences of a changing planet, including the spread of alien species, climate change and pollinator declines. Most of these topics represent new frontiers whose exploration has just begun.

Conclusions

Competition for pollination has served as a model for the integration of ecological and evolutionary perspectives in the study of species interactions. Its study has elucidated both obvious and more subtle mechanisms, and has documented a range of outcomes. However, the potential for this interaction to inform our understanding of both pure and applied aspects of pollination biology has only begun to be realized.Key words: Alien plants, climate change, competition for pollination, facilitation, mating system, mechanism, Lythrum, Mimulus, pollinator visitation, sexual function, invasive species, pollen loss     

Size-specific interaction patterns and size matching in a plant�Cpollinator interaction web

Background and Aims

Many recent studies show that plant–pollinator interaction webs exhibit consistent structural features such as long-tailed distributions of the degree of generalization, nestedness of interactions and asymmetric interaction dependencies. Recognition of these shared features has led to a variety of mechanistic attempts at explanation. Here it is hypothesized that beside size thresholds and species abundances, the frequency distribution of sizes (nectar depths and proboscis lengths) will play a key role in determining observed interaction patterns.

Methods

To test the influence of size distributions, a new network parameter is introduced: the degree of size matching between nectar depth and proboscis length. The observed degree of size matching in a Spanish plant–pollinator web was compared with the expected degree based on joint probability distributions, integrating size thresholds and abundance, and taking the sampling method into account.

Key Results

Nectar depths and proboscis lengths both exhibited right-skewed frequency distributions across species and individuals. Species-based size matching was equally close for plants, independent of nectar depth, but differed significantly for pollinators of dissimilar proboscis length. The observed patterns were predicted well by a model considering size distributions across species. Observed size matching was closer when relative abundances of species were included, especially for flowers with openly accessible nectar and pollinators with long proboscises, but was predicted somewhat less successfully by the model that included abundances.

Conclusions

The results suggest that in addition to size thresholds and species abundances, size distributions are important for understanding interaction patterns in plant–pollinator webs. It is likely that the understanding will be improved further by characterizing for entire communities how nectar production of flowers and energetic requirements of pollinators covary with size, and how sampling methods influence the observed interaction patterns.Key words: Plant–pollinator community, flower morphology, generalization, nectar, pollination network, body size, size matching, specialization     

Temperature-dependent development of the predatory mite <Emphasis Type="Italic">Cheyletus malaccensis</Emphasis> (Acari: Cheyletidae)

The effect of temperature on the development of immature stages of the predator Cheyletus malaccensis Oudemans, produced by either fertilized or virgin females, was studied at 17.5, 20, 25, 30, 32.5, and 35°C, 80 ± 5% relative humidity, in complete darkness, and fed Tyrophagus putrescentiae (Schrank). The population maintained at 15°C failed to reproduce. The thermal data obtained were used for the estimation of the thermal requirements (developmental thresholds, thermal constant, optimum temperature) of this predator by a linear and nonlinear model (Logan type I model). Upper and lower developmental thresholds ranged between 37.4–37.8 and 11.6–12.0°C, respectively. Optimum temperature for development was estimated at between 33.1 and 33.5°C. The thermal constant ranged between 238.1 and 312.5 degree-days. Based on the data of the total pre-imaginal period, immatures’ survival peaked at 25°C. Arrhenotokous parthenogenesis (haplodiploidy) is confirmed in the species: virgin females always produce males, whereas fertilized females give rise to offspring of both sexes. Survival of the immature stages and offspring sex ratio were not significantly influenced by temperature.     

Coupling Sequence-specific Recognition to DNA Modification

Enzymes that modify DNA are faced with significant challenges in specificity for both substrate binding and catalysis. We describe how single hydrogen bonds between M.HhaI, a DNA cytosine methyltransferase, and its DNA substrate regulate the positioning of a peptide loop which is ∼28 Å away. Stopped-flow fluorescence measurements of a tryptophan inserted into the loop provide real-time observations of conformational rearrangements. These long-range interactions that correlate with substrate binding and critically, enzyme turnover, will have broad application to enzyme specificity and drug design for this medically relevant class of enzymes.Sequence-specific modification of DNA is essential for nearly all forms of life and contributes to a myriad of biological processes including gene regulation, mismatch repair, host defense, DNA replication, and genetic imprinting. Methylation of cytosine and adenine bases is a key epigenetic process whereby phenotypic changes are inherited without altering the DNA sequence (1). The central role of the bacterial and mammalian S-adenosylmethionine (AdoMet)²-dependent DNA methyltransferases in virulence regulation and tumorigenesis, respectively, have led these enzymes to be validated targets for antibiotic and cancer therapies (2, 3). However, AdoMet-dependent enzymes catalyze diverse reactions, and the design of potent and selective DNA methyltransferase inhibitors is particularly challenging (4, 5). The design of drugs that bind outside the active site is a particularly attractive means of inhibition for enzymes with common cofactors like AdoMet because off-target inhibition often leads to toxicity (6). Unfortunately, robust methods to identify and characterize such critical binding sites distal from the active site have not been developed.DNA methyltransferases bind to a particular DNA sequence, stabilize the target base into an extrahelical position within the enzyme active site, and transfer the methyl moiety from AdoMet to the DNA (7). During this process, dramatic changes in the DNA structure such as bending, base flipping, or the intercalation of residues into the recognition sequence are often accompanied by large scale protein rearrangements (8). Here we characterized a specific conformational rearrangement of M.HhaI, a model DNA cytosine C⁵ methyltransferase with a cognate recognition sequence of 5′-GCGC-3′. Many structures of M.HhaI are available at high resolution including an ensemble of complexes with either cognate or nonspecific DNA (9, 10). Reorganization of an essential catalytic loop (residues 80–100) is regulated by sequence-specific protein-DNA interactions that occur ∼28 Å away from the catalytic loop (Fig. 1). Our work quantifies the importance of such distal communication in sequence-specific DNA modification and provides plausible structural mechanisms.Open in a separate windowFIGURE 1.Loop interactions in M.HhaI. A, two superimposed structures of M.HhaI are shown with the catalytic loop highlighted. Enzymes are in blue (open conformer) and red (closed conformer) with the cofactor in orange, the flipped cytosine in green, and position 1 of the DNA recognition sequence colored by atom along with Cys-81, Ile-86, and Arg-240. The large blue arrow shows the long-range structural communication between Arg-240 and the catalytic loop. B, close-up of the interactions between Arg-240, Ile-86, and position 1 of the recognition sequence. The flipped cytosine is in green. Removal of N² from the guanosine with an inosine base maintains near cognate loop motion while removal of O⁶ with a 2AP base has almost no loop motion.DNA-dependent positioning of the catalytic loop in M.HhaI was first observed crystallographically; cognate DNA stabilizes the loop-closed conformer, while nonspecific DNA leaves the loop in the open conformer (9, 10). Correct positioning of this loop is essential for catalysis because C81, the active site nucleophile that attacks the target cytosine base at the C⁶ position (supplemental Fig. S1), is ∼9.6 Å away in the loop-open conformer (Fig. 1A). Populating the closed conformer of the loop is essential for tight DNA binding and stabilizing the target cytosine that is flipped out of the DNA duplex (11–13). Using stopped-flow fluorescence spectroscopy to monitor the environment of tryptophan (Trp) residues inserted into the catalytic loop, we recently observed reorganization of this loop upon DNA binding in the absence of cofactor using the M.HhaI mutants W41F, W41F/K91W, and W41F/E94W (12). Loop positioning and the interconversion between the open and closed conformers, as determined from the intensities and rates of change in fluorescence signal are highly dependent on DNA sequence and confirm that cognate DNA stabilizes the loop-closed conformer whereas nonspecific DNA stabilizes the open conformer.In this study, W41F/K91W and W41F/E94W M.HhaI were preincubated with cognate (COG), non-cognate (NC), or nonspecific (NS) DNA and mixed with cofactor or cofactor product, AdoMet and S-adenosylhomocysteine (AdoHcy), respectively, in a stopped-flow apparatus. Differences in observed fluorescence intensity are indicative of shifts in the populations of the various loop conformers; no observable fluorescence change suggests no significant change in population and thus, essentially no loop positioning to the closed conformer. Non-cognate and cognate sequences are nearly identical but differ by a single base change, whereas the nonspecific DNA substrate has no similarity to the cognate sequence. As a methyltransferase searches for the cognate site within a genome, it must encounter both non-cognate and nonspecific DNA sequences and be able to distinguish these from the cognate sequence. We examine both binding, using the cofactor product AdoHcy, and catalysis, using the AdoMet cofactor of M.HhaI, with these various DNA substrates.     

Comparative Genomics of Multiple Strains of Pseudomonas cannabina pv. alisalensis,a Potential Model Pathogen of Both Monocots and Dicots

Comparative genomics of closely related pathogens that differ in host range can provide insights into mechanisms of host-pathogen interactions and host adaptation. Furthermore, sequencing of multiple strains with the same host range reveals information concerning pathogen diversity and the molecular basis of virulence. Here we present a comparative analysis of draft genome sequences for four strains of Pseudomonas cannabina pathovar alisalensis (Pcal), which is pathogenic on a range of monocotyledonous and dicotyledonous plants. These draft genome sequences provide a foundation for understanding host range evolution across the monocot-dicot divide. Like other phytopathogenic pseudomonads, Pcal strains harboured a hrp/hrc gene cluster that codes for a type III secretion system. Phylogenetic analysis based on the hrp/hrc cluster genes/proteins, suggests localized recombination and functional divergence within the hrp/hrc cluster. Despite significant conservation of overall genetic content across Pcal genomes, comparison of type III effector repertoires reinforced previous molecular data suggesting the existence of two distinct lineages within this pathovar. Furthermore, all Pcal strains analyzed harbored two distinct genomic islands predicted to code for type VI secretion systems (T6SSs). While one of these systems was orthologous to known P. syringae T6SSs, the other more closely resembled a T6SS found within P. aeruginosa. In summary, our study provides a foundation to unravel Pcal adaptation to both monocot and dicot hosts and provides genetic insights into the mechanisms underlying pathogenicity.     

Behavior and food consumption pattern of the population exposed in 1949�C1962 to fallout from Semipalatinsk nuclear test site in Kazakhstan

The relationship between radiation exposure from nuclear weapons testing fallout and thyroid disease in a group of 2,994 subjects has been the subject of study by the US National Cancer Institute. In that study, radiation doses to the thyroid were estimated for residents of villages in Kazakhstan possibly exposed to deposition of radioactive fallout from nuclear testing conducted by the Soviet Union at the Semipalatinsk Nuclear Test Site in Kazakhstan between 1949 and 1962. The study subjects included individuals of both Kazakh and Russian origin who were exposed during childhood and adolescence. An initial dose reconstruction used for the risk analysis of Land et al. (Radiat Res 169:373?C383, 2008) was based on individual information collected from basic questionnaires administered to the study population in 1998. However, because data on several key questions for accurately estimating doses were not obtained from the 1998 questionnaires, it was decided to conduct a second data collection campaign in 2007. Due to the many years elapsed since exposure, a well-developed strategy was necessary to encourage accurate memory recall. In our recent study, a focus group interview data collection methodology was used to collect historical behavioral and food consumption data. The data collection in 2007 involved interviews conducted within four-eight-person focus groups (three groups of women and one group of men) in each of four exposed villages where thyroid disease screening was conducted in 1998. Population-based data on relevant childhood behaviors including time spent in- and outdoors and consumption rates of milk and other dairy products were collected from women??s groups. The data were collected for five age groups of children and adolescents ranging from less than 1?year of age to 21?years of age. Dairy products considered included fresh milk and other products from cows, goats, mares, and sheep. Men??s focus group interviews pertained to construction materials of houses and schools, and animal grazing patterns and feeding practices. The response data collected are useful for improving estimates of thyroid radiation dose estimates for the subjects of an ongoing epidemiological study.     

Introduction of Aromatic Ring-Containing Substituents in Cyclic Nucleotides Is Associated with Inhibition of Toxin Uptake by the Hepatocyte Transporters OATP 1B1 and 1B3

Analogs of the cyclic nucleotides cAMP and cGMP have been extensively used to mimic or modulate cellular events mediated by protein kinase A (PKA), Exchange protein directly activated by cAMP (Epac), or protein kinase G (PKG). We report here that some of the most commonly used cyclic nucleotide analogs inhibit transmembrane transport mediated by the liver specific organic anion transporter peptides OATP1B1 and OATP1B3, unrelated to actions on Epac, PKA or PKG. Several cAMP analogs, particularly with 8-pCPT-substitution, inhibited nodularin (Nod) induced primary rat hepatocyte apoptosis. Inhibition was not mediated by PKA or Epac, since increased endogenous cAMP, and some strong PKA- or Epac-activating analogs failed to protect cells against Nod induced apoptosis. The cAMP analogs inhibiting Nod induced hepatocyte apoptosis also reduced accumulation of radiolabeled Nod or cholic acid in primary rat hepatocytes. They also inhibited Nod induced apoptosis in HEK293 cells with enforced expression of OATP1B1 or 1B3, responsible for Nod transport into cells. Similar results were found with adenosine analogs, disconnecting the inhibitory effect of certain cAMP analogs from PKA or Epac. The most potent inhibitors were 8-pCPT-6-Phe-cAMP and 8-pCPT-2′-O-Me-cAMP, whereas analogs like 6-MB-cAMP or 8-Br-cAMP did not inhibit Nod uptake. This suggests that the addition of aromatic ring-containing substituents like the chloro-phenyl-thio group to the purines of cyclic nucleotides increases their ability to inhibit the OATP-mediated transport. Taken together, our data show that aromatic ring substituents can add unwanted effects to cyclic nucleotides, and that such nucleotide analogs must be used with care, particularly when working with cells expressing OATP1B1/1B3, like hepatocytes, or intact animals where hepatic metabolism can be an issue, as well as certain cancer cells. On the other hand, cAMP analogs with substituents like bromo, monobutyryl were non-inhibitory, and could be considered an alternative when working with cells expressing OATP1 family members.     

Cdt1 is differentially targeted for degradation by anticancer chemotherapeutic drugs

Background

Maintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent re-replication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis.

Methodology/Principal Finding

In the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA.

Conclusion/Significance

Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy.