Wilma L. Lingle

Regulatable endogenous production of cytokinins up to toxic levels in transgenic plants and plant tissues

The effects of expressing a chimeric gene consisting of a soybean heat shock gene promoter and a sequence that encodes an enzyme catalyzing the synthesis of a potent phytohormone, the cytokinin iPMP, have been analyzed in transgenic tobacco plants. The production of cytokinin endogenously produced several effects previously undocumented. The differentiation of shoots independent of exogenous cytokinin from heat-treated transgenic plant leaf explants demonstrates that long-term heat treatments do not interfere with complex developmental processes. This extends the potential usefulness of heat shock gene promoters to conditionally express genes during windows of development that span several weeks.

Visualization of bioluminescence as a marker of gene expression in rhizobium-infected soybean root nodules

The linked structural genes lux A and lux B, encoding bacterial luciferase of a marine bacterium Vibrio harveyi, were fused with the nitrogenase nifD promoter from Bradyrhizobium japonicum and with the P1 promoter of pBR322. Both fusions were integrated into the B. japonicum chromosome by site-specific recombination. Soybean roots infected with the two types of rhizobium transconjugants formed nitrogen-fixing nodules that produced bright blue-green light. Cells containing the P1 promoter/lux AB fusion resulted in continuously expressed bioluminescence in both free-living rhizobium and in nodule bacteriods. However, when under control of the nifD promoter, luciferase activity was found only in introgen-fixing nodules. Light emission from bacteroids allowed us to visualize and to photograph nodules expressing this marker gene fusion in vivo at various levels of resolution, including within single, living plant cells. Localization of host cells containing nitrogen-fixing bacteroids within nodule tissue was accomplished using low-light video microscopy aided by realtime image processing techniques developed specifically to enhance extreme low-level luminescent images.

The effects of lethal heat shock on nonadapted and thermotolerant root cells of Glycine max

Abstract Exposure of nonadapted soybean seedlings to heat shock at 45°C results in death of the primary root. Thermotolerance, the acquired resistance to lethal heat shock, can be induced by successive treatments at 40 and 28°C. To better characterize the changes occurring during the acquisition of thermotolerance, transmission electron microscopy was used to examine the effects of permissive and lethal heat shock on nonadapted and thermotolerant soybean root cells. While the integrity of nonadapted cortical cells was completely lost during lethal heat shock, thermotolerant cells remained intact. All membrane-bound structures observed in control and permissively heat-shocked cells were observed in thermotolerant cells with the exception of dictyosomes. Structural changes were also evident in the nucleoplasm, nucleoli, and mitochondria after heat shock. These results are discussed in terms of previously described observations on the physiology and molecular biology of thermotolerance.

SPECTRAL ANALYSIS OF BIOLUMINESCENCE OF PANELLUS STYPTICUS

Corrected in vivo bioluminescence emission spectra have been obtained from monokaryotic and dikaryotic cultures and mature fruiting bodies of Pannellus stypticus. The wavelength of maximum bioluminescence was found at 525 nm. The bioluminescence emission was shifted to longer wavelengths, with a maximum at 528 nm, in basidiocarps that were deeply pigmented. The wavelengths of maximum bioluminescence of a number of fungi reported in the literature were determined to be from uncorrected spectra that legitimately cannot be compared with each other.

Endolithic thraustochytrid marine fungi from planted shell fragments

A thraustochytrid zoosporic fungus with characteristics of the genus Schizochytrium is a common member of an assemblage of different endolithic marine microorganisms found in carbonate shell fragments. We used clean shell fragments as bait in various subtidal marine locations on the coasts of Maine, Georgia and Jamaica. Heterotrophs (including fungi) were selected by covering each bait panel with a stainless steel shade that allowed sea water to circulate easily over the shell fragments. Shell fragments were recovered after six months. Details of the cytology of the endolithic organisms were revealed by a technique which produced a resin cast of the endoliths, or their traces, after fixed and embedded shells were decalcified. Scanning electron microscope observations were of the resin casts and transmission electron microscope observations were of sections of reembedded resin casts. Thraustochytrids were particularly abundant in fragments of mussel shells, which have more organic matrix than clam or oyster shells. Within the shells the thraustochytrids formed unusual elongated, tapered, and sometimes branched thalli that are divided into many vegetative cells, a characteristic feature of Schizochytrium. The cell nearest the shell surface typically was a zoosporangium containing biflagellate zoospores. Ultrastructural evidence for the thraustochytrid nature of the endoliths included a layered wall of dictyosome-derived thin scales and the production of an ectoplasmic network from bothrosomes on the cell surface. This role of thraustochytrid fungi in bioerosion is new and unexpected.

Localization of bioluminescent tissues during basidiocarp development in Panellus stypticus

Monokaryotic and dikaryotic mycelial cultures of Panellus stypticus are bioluminescent when grown on solid medium. Fruiting body primordia on fallen hardwood have minimal bioluminescence (≤1×10 8 photons emitted per second per primordium). Bioluminescence emission was observed to increase 10- to 50-fold during basidiocarp development. Distribution of bioluminescent tissues in the mature fruiting body is not uniform, but is restricted to the edge of radial and transverse lamellae, junction of lamellae with stipe and pileus margin (...)

PRELIMINARY ANALYSIS OF GENETIC COMPLEMENTATION OF BIOLUMINESCENCE IN PANELLUS STYPTICUS ISOLATED FROM PINE AND HARDWOOD

Inheritance of bioluminescence in North American populations of Panellus stypticus was analyzed using a set of five nonbioluminescent monokaryons and one bioluminescent monokaryon obtained from a basidiocarp of P. stypticus collected on pine wood. These were crossed with two nonbioluminescent and two bioluminescent monokaryons obtained from basidiocarps collected on hardwood in four geographically separate locations. Sexual compatibility of the geographically separate isolates indicated they were of the same biological species. Changing levels of bioluminescence intensities over time in the resulting dikaryotic cultures indicated that bioluminescence may be under stage-specific regulation. Inheritance of bioluminescence was analyzed according to the actual versus the expected maximal levels of bioluminescence intensity in dikaryons having at least one bioluminescent monokaryotic component. Bioluminescence was a dominant feature among the isolates tested. Genetic complementation groups established for dikaryons with two nonbioluminescent monokaryotic components suggested that three separate naturally occurring mutations can lead to lack of bioluminescence. Inter-allelic complementation may be involved in the expression of bioluminescence in a dikaryon resulting from a cross of two nonbioluminescent monokaryons. No significant differences among dikaryons were found in the characterization of bioluminescence emission spectra regardless of monokaryon composition.

Observations on tubular endoplasmic reticulum in the aquatic fungiBlastocladiella emersonii, Blastocladiella britannica, andCatenaria anguillulae

Abstract Tubular smooth endoplasmic reticulum membranes have been found in the cytoplasm of Blastocladiella emersonii, Blastocladiella britannica , and Catenaria anguillulae . These structures are formed and accumulate during the growth phase and are associated with cisternae of rough endoplasmic reticulum during sporangium formation. It is within the cisternae of rough of endoplasmic reticulum that the protein precursors of gamma particles are formed. It is probable that the tubular endomembranes represent a reservoir of membrane material that is formed during the growth phase and is converted into other endomembranes during sporogenesis.