COMPUTER PROGRAM FOR THREE-DIMENSIONAL RECONSTRUCTIONS AND NUMERICAL ANALYSES OF PLANT ORGANS FROM SERIAL SECTIONS

A computer program (SECTION) is presented which allows for the three-dimensional reconstruction of serial cross sections through biological materials. The program provides a numerical analysis of perimeter length and transverse area of each anatomical feature designated in a cross section, as well as surface area and volume computations for features that pass from one section to another. In addition to rotating and tilting the 3-D reconstruction in any desired orientation, the program has editing capabilities which allow different combinations of anatomical features to be shown in neutral gray outline or interconnected by colored lines.     

AERODYNAMICS OF EPHEDRA TRIFURCA: I. POLLEN GRAIN VELOCITY FIELDS AROUND STEMS BEARING OVULES

The behavior of pollen grains within the airspace around Ephedra trifurca is described. Vectoral analyses of pollen grains moving around stems and ovules indicate a complex pattern of directional and magnitudinal changes in trajectories that can be related to the geometries of surfaces that obstruct airflow. Pollen grains, passing around cylindrical stems oriented normal to the direction of ambient airflow, are deflected in circumrotating non-laminar flow-patterns. Stems tilted downwind deflect pollen grains into trajectories along leeward surfaces of stems. These trajectories travel acropetally in a spiraling pattern and may intersect airflow patterns created around and by ovules. Computer analyses of pollen motion in the vicinity of ovules indicate that pollen vector-direction is highly canalized and directed toward micropyles. Within the immediate vicinity of micropyles, which produce pollination droplets, analyses indicate that the magnitudinal variance of pollen grain vectors is high (spanning three orders of magnitude). This variance coincides with dramatic changes in the local Reynolds numbers, resulting in a localized region around the micropyle in which neither viscous nor inertial forces predominate. Based on additional aerodynamics parameters (vector curl, vector-divergence, and vector curl-differential) it is shown that the region around the pollination-droplet is characteristically a “pollen sink” (pollen grains collect in this airspace) in which abrupt changes can occur in the angular momenta of airborne pollen grains. These aerodynamic analyses suggest that the morphology of ovules and the stems to which they are attached facilitates pollen capture by creating an aerodynamic “singularity” (= a unique region) around the pollination-droplet.     

CONIFER OVULATE CONE MORPHOLOGY: IMPLICATIONS ON POLLEN IMPACTION PATTERNS

Empirically determined patterns of pollen impaction on the surfaces of pine ovulate cones are correlated with regions of nonlaminar flow created by the spatial arrangement and morphology (aspect ratios) of scale-bract complexes. Results from the serial discharge of pollen, upwind of ovulate cones, indicate that ovules on ovulate cones are preferentially impacted by pollen from their own species. Analyses indicate that while aerodynamic factors dominate the entrapment of pollen by ovulate cones, other factors such as pollen impaction-rebound and rebound-reentrainment are significant. Surface characteristics in addition to the settling velocities of pollen may play important roles in determining pollination efficiency. Wind tunnel analyses of the aerodynamic effects of scale-bract arrangement and aspect ratios indicate that each complex behaves as an aerofoil, deflecting air eddies toward the micropylar ends of ovules. The ovulate cone geometry, as a whole, deflects unidirectional wind into cyclonic vortices around the cone axis, each scale-bract deflecting nonimpacted pollen along orthostichies and parastichies. The morphology of the typical conifer ovulate cone is interpreted as a structure that optimizes anemophilous reproduction.     

Characterization of phase separation in film forming biopolymer mixtures

Enhanced, tailor-made films can be achieved by combining the good gas barrier of the hydrophilic high amylose maize starch (hylon) with the water resistance of the hydrophobic protein zein. Two polymers are not always miscible in solution, and the phase separation behavior of the mixture is therefore important for the final film structure and its properties. Phase separation of a mixture of these two biopolymers was induced either by cooling, which was observed as growing droplets of the hylon phase which in some cases also formed small aggregates, or by solvent evaporation and studied in real-time in a confocal laser scanning microscope. Solvent evaporation had a much stronger effect on phase separation. During the early stage of phase separation, hylon formed large aggregates and subsequently smaller droplets coalesced with other droplets or large hylon aggregates. The later part of the separation seemed to take place through spinodal decomposition.     

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

The Arabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1, expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp-167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro target of AtPDK1. Our data clearly show that Asp-167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident.     

Biomass partitioning and leaf N,P - stoichiometry: comparisons between tree and herbaceous current-year shoots

We compare the biomass partitioning patterns and the nitrogen/phosphorus (N,P) stoichiometry of the current-year shoots of tree and herbaceous species and ask whether they scale in the same ways. Our analyses indicate that few statistically significant differences exist between the shoot biomass partitioning patterns of the two functional species-groups. In contrast, statistically significant N,P - stoichiometric differences exist between the two functional groups. Across all species, dry leaf mass scales nearly as the square of basal stem diameter and isometrically with respect to dry stem mass. However, total leaf N scales as the 1.37-power and as the 1.09-power of total leaf P across herbaceous and tree shoots, respectively. Therefore, tree shoots can be viewed as populations of herbs elevated by their older, woody herbaceous cohorts. However, tree leaf stoichiometry cannot be modelled in terms of herbaceous N,P - leaf stoichiometry.     

Higher level phylogeny of Satyrinae butterflies (Lepidoptera: Nymphalidae) based on DNA sequence data

We have inferred the first empirically supported hypothesis of relationships for the cosmopolitan butterfly subfamily Satyrinae. We used 3090 base pairs of DNA from the mitochondrial gene COI and the nuclear genes EF-1alpha and wingless for 165 Satyrinae taxa representing 4 tribes and 15 subtribes, and 26 outgroups, in order to test the monophyly of the subfamily and elucidate phylogenetic relationships of its major lineages. In a combined analysis, the three gene regions supported an almost fully resolved topology, which recovered Satyrinae as polyphyletic, and revealed that the current classification of suprageneric taxa within the subfamily is comprised almost completely of unnatural assemblages. The most noteworthy findings are that Manataria is closely related to Melanitini; Palaeonympha belongs to Euptychiina; Oressinoma, Orsotriaena and Coenonympha group with the Hypocystina; Miller's (1968). Parargina is polyphyletic and its components group with multiple distantly related lineages; and the subtribes Elymniina and Zetherina fall outside the Satyrinae. The three gene regions used in a combined analysis prove to be very effective in resolving relationships of Satyrinae at the subtribal and tribal levels. Further sampling of the taxa closely related to Satyrinae, as well as more extensive sampling of genera within the tribes and subtribes for this group will be critical to test the monophyly of the subfamily and establish a stronger basis for future biogeographical and evolutionary studies.     

Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants

Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.