Orchid phylogenomics and multiple drivers of their extraordinary diversification

Orchids are the most diverse family of angiosperms, with over 25 000 species, more than mammals, birds and reptiles combined. Tests of hypotheses to account for such diversity have been stymied by the lack of a fully resolved broad-scale phylogeny. Here, we provide such a phylogeny, based on 75 chloroplast genes for 39 species representing all orchid subfamilies and 16 of 17 tribes, time-calibrated against 17 angiosperm fossils. A supermatrix analysis places an additional 144 species based on three plastid genes. Orchids appear to have arisen roughly 112 million years ago (Mya); the subfamilies Orchidoideae and Epidendroideae diverged from each other at the end of the Cretaceous; and the eight tribes and three previously unplaced subtribes of the upper epidendroids diverged rapidly from each other between 37.9 and 30.8 Mya. Orchids appear to have undergone one significant acceleration of net species diversification in the orchidoids, and two accelerations and one deceleration in the upper epidendroids. Consistent with theory, such accelerations were correlated with the evolution of pollinia, the epiphytic habit, CAM photosynthesis, tropical distribution (especially in extensive cordilleras), and pollination via Lepidoptera or euglossine bees. Deceit pollination appears to have elevated the number of orchid species by one-half but not via acceleration of the rate of net diversification. The highest rate of net species diversification within the orchids (0.382 sp sp⁻¹ My⁻¹) is 6.8 times that at the Asparagales crown.     

Filamentous Proteins from Plant Sieve Tubes

PROTEIN filaments are characteristic structural components of the assimilatory conducting elements of angiosperm plants (“P protein” of Cronshaw and Esau¹). We have isolated filamentous structures from the phloem exudate of cut cucurbit stems². The presence of the filaments could be clearly demonstrated after negative staining with the electron microscope.     

Comparison of techniques for the measurement of skin temperature during exercise in a hot,humid environment

Exercising or working in a hot, humid environment can results in the onset of heat-related illness when an individual''s temperature is not carefully monitored. The purpose of the present study was to compare three techniques (data loggers, thermal imaging, and wired electrodes) for the measurement of peripheral (bicep) and central (abdominal) skin temperature. Young men and women (N = 30) were recruited to complete the present study. The three skin temperature measurements were made at 0 and every 10-min during 40-min (60% VO₂max) of cycling in a hot (39±2°C), humid (45±5% RH) environment. Data was statistically analyzed using the Bland-Altman method and correlation analysis. For abdominal skin temperature, the Bland-Altman limits of agreement indicated that data loggers (1.5) were a better index of wired than was thermal imaging (3.5), For the bicep skin temperature the limits of agreement was similar between data loggers (1.9) and thermal (1.9), suggesting the both were suitable measurements. We also found that when skin temperature exceeded 35°C, we observed progressively better prediction between data loggers, thermal imaging, and wired skin sensors. This report describes the potential for the use of data loggers and thermal imaging to be used as alternative measures of skin temperature in exercising, human subjects.     

Plant community and phylogenetic shifts in acid seep springs over 49 years following Microstegium vimineum invasion

Plant Ecology - Community composition and diversity can change following exotic invasion. We tested the extent to which acid seep spring communities in southern Illinois had changed in composition...     

Hi-Fi transmission of periodic signals amid cell-to-cell variability

Since information in intracellular calcium signaling is often frequency encoded, it is physiologically critical and experimentally useful to have reliable, convenient, and non-invasive methods to entrain it. Because of cell-to-cell variability, synchronization of intracellular signaling across a population of genetically identical cells can still be difficult to achieve. For intrinsically oscillatory signaling pathways, such as calcium, upon continuous stimulation, cell-to-cell variability is manifested as differences in intracellular response frequencies. Even with entrainment using periodic stimulation, cell-to-cell variability is manifested as differences in the fidelity with which extracellular inputs are converted into intracellular signals. Here we present a combined theoretical and experimental analysis that shows how to appropriately balance stimulation strength, duration, and rest intervals to achieve entrainment with high fidelity stimulation-to-response ratios for G-protein-coupled receptor-triggered intracellular calcium oscillations. We further demonstrate that stimulation parameters that give high fidelity entrainment are significantly altered upon changes in intracellular enzyme levels and cell surface receptor levels. Theoretical analysis suggests that, at key threshold values, even small changes in these protein concentrations or activities can result in precipitous changes in entrainment fidelity, with implications for pathophysiology.     

Effects of taxon sampling and tree reconstruction methods on phylodiversity metrics

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Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues

Hydrolysis of fluorescent GTP analogues BODIPY FL guanosine 5 '-O-(thiotriphosphate) (BGTPgammaS) and BODIPY FL GTP (BGTP) by Galpha(i1) and Galpha was characterized using on-line capillary electrophoresis (o) laser-induced fluorescence assays in order that changes in sub-strate, substrate-enzyme complex, and product could be monitored separately. Apparent k values (V /[E]) (max cat) steady-state and K(m) values were determined from assays for each substrate-protein pair. When BGTP was the substrate, maximum turnover numbers for Galpha and Galpha(i1) were 8.3 +/- 1 x 10(-3) and 3.0 +/- 0.2 x 10(-2) s(-1), respectively, and K(m) values were 120 +/- 60 and 940 +/- 160 nm. Assays with BGTPgammaS yielded maximum turnover numbers of 1.6 +/- 0.1 x 10(-4) and 5.5 +/- 0.3 x 10(-4) s(-1) for Galpha and Galpha(i1); K(m) values were 14 (o)(+/-)8 and 87 +/- 22 nm. Acceleration of Galpha GTPase activity by regulators of G protein signaling (RGS) was demonstrated in both steady-state and pseudo-single-turnover assay formats with BGTP. Nanomolar RGS increased the rate of enzyme product formation (BODIPY(R) FL GDP (BGDP)) by 117-213% under steady-state conditions and accelerated the rate of G protein-BGTP complex decay by 199 -778% in pseudo-single-turnover assays. Stimulation of GTPase activity by RGS proteins was inhibited 38-81% by 40 mum YJ34, a previously reported peptide RGS inhibitor. Taken together, these results illustrate that Galpha subunits utilize BGTP as a substrate similarly to GTP, making BGTP a useful fluorescent indicator of G protein activity. The unexpected levels of BGTPgammaS hydrolysis detected suggest that caution should be used when interpreting data from fluorescence assays with this probe.     

Subcellular distribution of alpha 2-adrenergic receptors, pertussis-toxin substrate and adenylate cyclase in human platelets.

The subcellular distribution of the alpha 2-adrenergic receptor, pertussis-toxin substrates (Gi, the inhibitory G-protein) and adenylate cyclase was determined in human platelets. The alpha 2-adrenergic receptor and pertussis-toxin substrate activity codistribute with surface membranes identified by a novel fluorescent-lectin method. The platelet granule fractions did not contain detectable Gi. Only 2-4% of the total pertussis-toxin substrate activity appears in soluble fractions, and this amount was not increased upon addition of purified beta gamma units or after pretreatment of platelets with adrenaline. There is no evidence for compartmentation of the alpha 2-adrenergic receptor or Gi to account for the low-affinity component of agonist binding to the alpha 2-adrenergic receptor in human platelet membranes. Translocation of Gi from plasma membrane to platelet cytosol or granules does not appear to play any significant role in the mechanism of alpha 2-receptor-mediated platelet activation.     

NMR structure of the second intracellular loop of the alpha 2A adrenergic receptor: evidence for a novel cytoplasmic helix

A major, unresolved question in signal transduction by G protein coupled receptors (GPCRs) is to understand how, at atomic resolution, a GPCR activates a G protein. A step toward answering this question was made with the determination of the high-resolution structure of rhodopsin; we now know the intramolecular interactions that characterize the resting conformation of a GPCR. To what degree does this structure represent a structural paradigm for other GPCRs, especially at the cytoplasmic surface where GPCR-G protein interaction occurs and where the sequence homology is low among GPCRs? To address this question, we performed NMR studies on approximately 35-residue-long peptides including the critical second intracellular loop (i2) of the alpha 2A adrenergic receptor (AR) and of rhodopsin. To stabilize the secondary structure of the peptide termini, 4-12 residues from the adjacent transmembrane helices were included and structures determined in dodecylphosphocholine micelles. We also characterized the effects on an alpha 2A AR peptide of a D130I mutation in the conserved DRY motif. Our results show that in contrast to the L-shaped loop in the i2 of rhodopsin, the i2 of the alpha 2A AR is predominantly helical, supporting the hypothesis that there is structural diversity within GPCR intracellular loops. The D130I mutation subtly modulates the helical structure. The spacing of nonpolar residues in i2 with helical periodicity is a predictor of helical versus loop structure. These data should lead to more accurate models of the intracellular surface of GPCRs and of receptor-mediated G protein activation.