Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda)

Biological ageing is connected to life history variation across ecological scales and informs a basic understanding of age-related declines in organismal function. Altered DNA methylation dynamics are a conserved aspect of biological ageing and have recently been modelled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic ageing, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe ageing in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 6% of its maximum lifespan. Although patterns of CHH-methylation showed little association with age, both CpG and CHG-methylation contexts were strongly associated with ageing, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological ageing in plants.     

Comparisons of biomass,water use efficiency and water use strategies across five genomic groups of Populus and its hybrids

Genetic improvement and hybridization in the Populus genus have led to the development of genotypes exhibiting fast growth, high rooting ability and disease resistance. However, while large biomass production is important for bioenergy crops, efficient use of resources including water is also important in sites lacking irrigation and for maintaining ecosystem water availability. In addition, comparison of water use strategies across a range of growth rates and genetic variability can elucidate whether certain strategies are shared among the fastest growing and/or most water use efficient genotypes. We estimated tree water use throughout the second growing season via sapflow sensors of 48 genotypes from five Populus taxa; P. deltoides W. Bartram ex Marshall × P. deltoides (D × D), P. deltoides × P. maximowiczii A. Henry (D × M), P. deltoides × P. nigra L. (D × N), P. deltoides × P. trichocarpa Torr. & Gray (D × T) and P. trichocarpa × P. deltoides (T × D) and calculated average canopy stomatal conductance (G_S). We regressed G_S and atmospheric vapor pressure deficit (VPD) wherein the slope of the relationship represents stomatal sensitivity to VPD. At the end of the second growing season, trees were harvested, and their dry woody biomass was used to calculate whole tree water use efficiency (WUE_T). We found that D × D and D × M genotypes exhibited differing water use strategies with D × D genotypes exhibiting high stomatal sensitivity while retaining leaves while D × M genotypes lost leaf area throughout the growing season but exhibited low stomatal sensitivity. Across measured taxa, biomass growth was positively correlated with WUE_T, and genotypes representing each measured taxa except D × N and T × D had high 2-year dry biomass of above 6 kg/tree. Overall, these data can be used to select Populus genotypes that combine high biomass growth with stomatal sensitivity and WUE_T to limit the negative impacts of bioenergy plantations on ecosystem water resources.     

COMPONENTS OF PHENOTYPIC SELECTION: POLLEN EXPORT AND FLOWER COROLLA WIDTH IN IPOMOPSIS AGGREGATA

In the hummingbird-pollinated herb Ipomopsis aggregata, selection through male function during pollination favors wide corolla tubes. We explored the mechanisms behind this selection, using phenotypic selection analysis to compare effects of corolla width on two components of male pollination success, pollinator visit rate and pollen exported per visit. During single visits by captive hummingbirds, flowers with wider corollas exported more pollen, and more dye used as a pollen analogue, to stigmas of recipient flowers. Corolla width was less strongly related to visit rate in the field, and had no direct effect on visit rate after nectar production and corolla length were controlled for. Moreover, the phenotypic selection differential was 80% higher for the effect on pollen exported per visit, suggesting that this is the more important mechanism of selection.     

Identification of cDNAs encoding isoprenylated proteins

Isoprenylated proteins are involved in signal transduction, control of cell growth and differentiation, organization of the nuclear lamina and cytoskeleton, and vesicle sorting. The isoprenoid moiety facilitates the interaction of these proteins with membranes and/or other proteins. However, many isoprenylated proteins remain unidentified. A method is described for identifying novel and known cDNAs encoding isoprenylated proteins. Sufficient details of the screening procedure are given so that this method may be easily used to identify cDNAs encoding other covalently modified proteins or proteins possessing high affinity ligand binding sites.     

The Second Messenger, Cyclic AMP, Is Not Sufficient for Myelin Gene Induction in the Peripheral Nervous System

Abstract: The adenylyl cyclase-cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin-specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8–10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P₀ mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P₀ gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short- or long-term treatment with IBMX and forskolin in the transected nerve. A three-fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger-dependent stimuli when Schwann cells are non-myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath.     

Effects of wall shear stress and fluid recirculation on the localization of circulating monocytes in a three-dimensional flow model

There is a correlation between the location of early atherosclerotic lesions and the hemodynamic characteristics at those sites. Circulating monocytes are key cells in the pathogenesis of atherosclerotic plaques and localize at sites of atherogenesis. The hypothesis that the distribution of monocyte adhesion to the vascular wall is determined in part by hemodynamic factors was addressed by studying monocyte adhesion in an in vitro flow model in the absence of any biological activity in the model wall.

Suspensions of U937 cells were perfused (Re = 200) through an axisymmetric silicone flow model with a stenosis followed by a reverse step. The model provided spatially varying wall shear stress, flow separation and reattachment, and a three-dimensional flow pattern. The cell rolling velocity and adhesion rates were determined by analysis of videomicrographs. Wall shear stress was obtained by numerical solution of the equations of fluid motion. Cell adhesion patterns were also studied in the presence of chemotactic peptide gradients.

The cell rolling velocity varied linearly with wall shear stress. The adhesion rate tended to decrease with increasing local wall shear stress, but was also affected by the radial component of velocity and the dynamics of the recirculation region and flow reattachment. Adhesion was increased in the vicinity of chemotactic peptide sources downstream of the expansion site. Results with human monocytes were qualitatively similar to the U937 experiments.

Differences in the adhesion rates of U937 cells occurring solely as a function of the fluid dynamic properties of the flow field were clearly demonstrated in the absence of any biological activity in the model wall.     

Chaperone SecB: Conformational changes demonstrated by circular dichroism

The chaperone SecB, which is involved in protein export inEscherichia coli, is shown by circular dichroism measurements to contain a high content of-pleated sheets. Prediction of the secondary structure of SecB is in good agreement with the observed content of-sheet. In accordance with the previous studies in which changes in conformation were assessed indirectly [Randall (1992),Science 257, 241–245], here we show that the conformation of SecB changes with the concentration of salt in the milieu and also when SecB interacts with a peptide ligand.Abbreviations ANS 1-anilino-naphthalene-8-sulfonate - CD circular dichroism - NMR nuclear magnetic resonance - CCA convex constraint analysis     

Determination of the binding frame of the chaperone SecB within the physiological ligand oligopeptide-binding protein.

Chaperone proteins demonstrate the paradoxical ability to bind ligands rapidly and with high affinity but with no apparent sequence specificity. To learn more about this singular property, we have mapped the binding frame of the chaperone SecB from E. coli on the oligopeptide-binding protein. Similar studies performed on the maltose-binding and galactose-binding proteins revealed centrally positioned binding frames of approximately 160 aminoacyl residues. The work described here shows that OppA, which is significantly longer than the previously studied ligands, has a binding frame that covers 460 amino acids, nearly the entire length of the protein. We propose modes of binding to account for the data.