A paragenetic perspective on integration of RNA silencing into the epigenome and its role in the biology of higher plants.

We describe features of RNA silencing and associated epigenetic imprints that illustrate potential roles for RNA interference (RNAi) in maintenance and transmission of epigenetic states between cells, throughout a plant, and perhaps even across sexual generations. Three types of transgenes can trigger RNAi of homologous endogenous plant genes: (1) "sense" transgenes that overexpress translatable transcripts, (2) inverted repeat (IR) transgenes that produce double-stranded RNA (dsRNA), and (3) antisense transgenes. Each mode of RNAi produces a different characteristic developmental silencing pattern. Single-copy transgenes are sufficient for sense-RNAi and antisense-RNAi, but not inverted repeat-RNAi. A single premature termination codon dramatically attenuates sense-RNAi, but it has no effect on antisense or inverted repeat-RNAi. We report here that antisense transgenes altered by removal of nonsense codons generate silencing patterns characteristic of sense-RNAi. Duplication of a sense overexpression transgene results in two types of epigenetic events: (1) complete loss of silencing and (2) altered developmental pattern of silencing. We also report that duplicating only the transgene promoter results in complete loss of silencing, whereas duplicating only transcribed sequences produces the second class, which are vein-based patterns. We infer that the latter class is due to systemic RNA silencing signals that interact with certain epigenetic states of the transgene to imprint it with information generated at a distance elsewhere in the plant.

Segregation distortion of T-DNA markers linked to the self-incompatibility (S) locus in Petunia hybrida.

In plants with a gametophytic self-incompatibility system the specificity of the pollen is determined by the haploid genotype at the self-incompatibility (S) locus. In certain crosses this can lead to the exclusion of half the gametes from the male parent carrying a particular S-allele. This leads to pronounced segregation distortion for any genetic markers that are linked to the S-locus. We have used this approach to identify T-DNA insertions carrying a maize transposable element that are linked to the S-locus of Petunia hybrida. A total of 83 T-DNA insertions were tested for segregation distortion of the selectable marker used during transformation with Agrobacterium. Segregation distortion was observed for 12 T-DNA insertions and at least 8 of these were shown to be in the same linkage group by intercrossing. This indicates that differential transmission of a single locus (S) is probably responsible for all of these examples of T-DNA segregation distortion. The identification of selectable markers in coupling with a functional S-allele will allow the preselection of recombination events around the S-locus in petunia. Our approach provides a general method for identifying transgenes that are linked to gametophytic self-incompatibility loci and provides an opportunity for transposon tagging of the petunia S-locus.

Reevaluating concepts of apical dominance and the control of axillary bud outgrowth.

A large amount of diversity of architectural form is found among flowering plants, and an important aspect of this diversity is the wide variation, ranging from simple to complex, found among branching patterns in plant shoot systems. Historically, the control of bud outgrowth has been attributed to the presence of a growing shoot apex. The term "apical dominance" is used to indicate that the shoot tip exerts an inhibitory control over proximal axillary buds. Through decapitation and/or hormone manipulation experiments, this inhibition has been attributed to the phytohormones auxin and cytokinin. Recent studies with mutants demonstrating increased branching indicate important additional roles for organs apart from those in the shoot tip and for signals other than cytokinin and auxin. This chapter provides a critical review of branching with an emphasis toward bud outgrowth in a developmental context. This review provides a detailed synopsis of physiological, genetic, and molecular studies and approaches for the investigation of branching regulation in plants.

Psl: a novel Spm-like transposable element from Petunia hybrida.

The identification of a spontaneous mutable Hf1 allele in Petunia hybrida provided an opportunity to isolate and characterize a novel transposable element. This 9.9 kb element has features in common with members of the Spm family, such as homologous terminal inverted repeats and a 3 bp target site direct duplication within the Hf1 gene. The element is named Petunia Spm-like (Psl). The footprints left by excising elements have been isolated from several germinal revertants and sequence analysis shows similarities to those left by other Spm family members. Southern analysis shows that the transposon is present at low copy number in the genome of different inbred lines and species of Petunia. The germinal excision frequency of Psl was 21-33% in outcross populations. The element appears to be very mobile somatically in the inbred line V26, with 38% of plants from an inbred population showing new Psl-hybridizing bands by Southern analysis. The high somatic and germinal excision frequency demonstrated by Psl suggests that this element may have utility for gene tagging in petunia.

Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences.

Flower pigmentation patterns were scored in 185 sense Chalcone synthase (Chs) transgenotes and 85 antisense Chs transgenotes; upon first flowering, 139 (75%) of sense transgenotes were found to be phenotypically altered, as were 70 (82%) of the antisense transgenotes. The observed patterns document the range of phenotypic variations that occur, as well as confirm and extend the finding that sense Chs constructs produce several types of morphology-based flower pigmentation patterns that antisense Chs constructs do not. Long-term monitoring for epigenetic variations in one population of 44 sense Chs transgenotes showed that 43 (98%) were capable of producing a cosuppression phenotype. The primary determinant of sense-specific patterns of cosuppression of Chs was found to be the repetitiveness and organization pattern of the transgene, not 'position effects' by, or 'readthrough' from, flanking plant DNA sequences. The degree of cosuppression observed in progeny of transgenotes carrying multiple, dispersed copies as compared to that observed with a single copy of the transgene suggests that sense cosuppression of Chs is subject to a transgene dosage effect.

Radionuclide assessment of the natural heart ejection fraction before and after LVAD implantation.

Complete pressure unloading of the ventricles can preserve ischemically damaged myocardium. Most clinical left heart assist device (LVAD) systems used after ischemic injury of the heart apply atrial cannulation which does not ensure pressure unloading. In order to assess the effect of the implantation of an intracorporeal LVAD on the function of the natural heart, we determined the ejection fraction (EF) in four male Holstein calves (90-105 kg) before and after insertion of a Cleveland Clinic pneumatic LVAD. A gated blood pool scan was obtained with a gamma camera after injection of 40 mCi Tc-labelled albumin. The animals were restrained in a sling to avoid movement artifacts. All animals showed a drop of 65 +/- 12% to 42 +/- 14% EF in the first postoperative (p.o.) week. Left ventricular output did not maintain sufficient blood pressure as assessed by pump-off tests. Systolic blood pressure dropped from 122 +/- 6.5 mm Hg to 81 +/- 6 mm Hg without pump support on the morning of the first p.o. day. Apical coring and possible restrained heart movement by the implanted LVAD may lead to impaired myocardial function that renders the individual LVAD dependent until adaptative corrections take place.

Alterations in cardiac function and cardiopulmonary blood volume in chronic sodium depletion in dogs.

1. Chronic sodium depletion in dogs is associated with a blunting of the pressor response to carotid occlusion. AFter section of the vagus nerves this pressor response reverts to normal although atropine is without effect, suggesting a possible role for increased activity of cardiopulmonary vagal afferents in suppressing sympathetic outflow. Since increases in central blood volume stimulate vagal afferents, cardiopulmonary haemodynamics were studied by radioisotope dilution before and after 3 and 4 weeks of dietary sodium restriction together with frusemide. 2. Sodium depletion was associated with significant decreases in cardiac output, ejection fraction and total blood volume; the cardiopulmonary blood volume increased but the change was not statistically significant (P < 0.2). These changes were accompanied by a significant increase in the ratio cardiopulmonary blood volume/total blood volume and a decrease in the ratio cardiac output/cardiopulmonary blood volume. 3. Results indicate that sodium depletion may be accompanied by a paradoxical translocation of blood to the cardiopulmonary region in part due to depressed ventricular performance.

Thallium-20 1 myocardial imaging in young adults with anomalous left coronary artery arising from the pulmonary artery.

Anomalous origin of the left coronary artery from the pulmonary artery (Bland-Garland-White syndrome) may produce myocardial ischemia, infarction, and frequently death in infancy. Some patients, however, develop satisfactory coronary artery collaterals and are relatively asymptomatic into adulthood. Very little is known about their myocardial perfusion patterns. We studied three young adults with this condition using stress thallium-20 1 myocardial imaging. Electrocardiograms in two patients demonstrated old anterolateral myocardial infarctions. Preoperative stress exercise tests were positive in all three patients. Marked perfusion abnormalities were found in the proximal anterolateral wall in all patients, and one patient also had a posterolateral defect. Postoperatively, all stress tests returned to normal. Thallium imaging demonstrated improvement in ischemic areas, but old scars persisted.

Adsorption of bacteria to roots as related to host specificity in the Rhizobium-clover symbiosis.

Quantitative microscope techniques were utilized to examine the adsorption of rhizobial cells to clover root hairs. Adsorption of cells of noninfective strains of Rhizobium trifolii or infective R. meliloti strains to clover root hairs was four to five times less than that of the infective R. trifolii strains. Attachment of the rod-shaped bacteria to clover root cells occurred in a polar, end-on fashion. Viable or heat-killed R. trifolii cells precoated with a clover lectin having 2-deoxyglucose specificity had increased adsorption to clover roots. Adsorption of bacteria to roots was not increased if the clover lectin was inactivated by heat or 2-deoxyglucose treatment prior to incubation with R. trifolii. Adsorption of R. trifolii to clover root hairs was inhibited by 2-deoxyglucose (30 mM) but not by 2-deoxygalactose or alpha-D-glucose. Adsorption of R. meliloti cells to alfalfa root hairs was not affected by 2-deoxyglucose at that concentration. These results suggest that expression of host specificity in the Rhizobium-clover symbiosis involves a preferential adsorption of infective cells to clover root hairs through a 2-deoxyglucose-sensitive receptor site.

Ultrastructure of Rhizobium-induced infection threads in clover root hairs.

Ultrastructural studies of Rhizobium-induced infection threads in clover root hairs show that the infection thread is initiated by an invagination process. Root hair wall growth is redirected at a localized point, resulting in the formation of an open pore. There is no direct penetration through the wall, and the bacteria remain extracellular within the root hair.