Clonal diversity in the rare Calamagrostis porteri ssp. insperata (Poaceae): comparative results for allozymes and random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers

Four populations of the rare, highly clonal grass Calamagrostis porteri ssp. insperata were examined using allozymes and the two polymerase chain reaction (PCR)-based markers, random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) bands. Only one of the 15 allozyme loci was variable and two alleles were detected, both of which were found in two populations, while only one genotype was detected in the other two populations. ISSR and RAPD markers detected more genotypes within populations than did allozymes. ISSR markers detected more diversity than RAPD markers in three of the four populations examined. In one population, no RAPD diversity was found whereas eight different genotypes were found among the 10 plants with ISSR markers. This diversity is present despite rare flowering, no documented occurrence of seed set in natural populations and very low seed set with experimental pollinations, all of which suggest that sexual reproduction rarely occurs. The subspecies is self-compatible, but seed initiation is lower in selfed ovules; also, there is high embryo abortion regardless of pollen source. Variation detected by RAPD and ISSR primers may reflect higher levels of sexual reproduction in the past, very rare sexual reproduction in extant populations, somatic mutations, or a combination of the three. Although the PCR-based markers identify several multilocus genotypes within populations, it is not known whether these all represent distinct genets generated by sexual reproduction or result from somatic mutations in the old, perennial and highly clonal plants.     

Lignification and Xylogenesis in Lactuca Pith Explants Cultured In Vitro in the Presence of Auxin and Cytokinin: A Role for Endogenous Ethylene

Lignification and xylogenesis were studied in lettuce (Lactucasativa L. cv. Romaine) pith parenchyma explants cultured ona Murashige and Skoog basal medium supplemented with indole-3-aceticacid, kinetin, and glucose. Lignin formation was observed tooccur in two distinct phases, one preceding xylem differentiation(days 0–3 of culture) and another coincident with maximalxylogenesis (days 4–7). The rate of soluble phenolic productionby these explants increased concomitant with the first phaseof lignification, then decreased during the second phase. Addition of silver, an ethylene antagonist, to the culture mediuminhibited the second phase of lignification and markedly reducedwall-bound peroxidase activity. Exogenous L-methionine, an ethyleneprecursor, completely reversed the inhibitory effect of silveron ligm6cation and wall-bound peroxidase activity. Silver increasedphenylalanine ammonia-lyase activity, but had no effect on solublephenolic production or soluble pcroxidase activity. These resultssuggest that ethylene may play a role in controlling lignificationduring xylogenesis by inducing wall-bound peroxidase activity. Key words: Auxin, Cytokinin, Ethylene, Xylogenesis, Lignification, Phenylalanine ammonia-lyase, Peroxidase     

Regional Ultrastructural Differences in Basal Laminae Isolated from the Starfish Pisaster ochraceus

The ultrastructure of different regions of the basal laminae isolated from 5-1/2-6 day-old embryos of the starfish, Pisaster ochraceus , has been described after fixation in the presence of anionic dyes. Isolated basal laminae from all regions of the embryo exhibit a lamina lucida and lamina densa. No lamina fibroreticularis is present. Instead, a coarse meshwork of thick densely stained and thinner intermediately stained fibers is embedded in the lamina densa and extends into the blastocoel forming the extracellular matrix. The coarse meshwork associated with the ectodermal basal lamina consists primarily of thick densely stained fibers with a small number of intermediate ones while that associated with the endodermal one contains much less densely stained material. These structures were morphologically identical to those found in control embryos. Examination of different regions of the endodermal basal lamina shows that the amount of dense material varies from region to region. These differences in dense material may reflect biochemical differences, particularly of proteoglycans, which could provide positional information to migrating mesenchyme cells.     

Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis

We investigated the use of multiplex polymerase chain reaction (FCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWWO), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWWO, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10* to 10* cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 μL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradarJve cells, at a lower detection limit of = > 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.     

Electrical Impedance Studies on Potato and Alfalfa Tissue

Small pulses of alternating current have been found to be conductedchiefly in the channels of the cell walls of tubers of potato(Solatium tuberosum), the roots and stems of alfalfa (Medicagosativa), and similar plant tissue. Impedance measurements mayusefully detect the state of water relations in these channelswhen the frequency of the measuring A.C. is low (around 60 c/s),when chlori-dized silver probes are used and when the cell-wallresistance is low compared to the cell membranes in series.Activation energies have been calculated for the conductivityof these tissues. Over the temperature and soil moisture rangesinvestigated, the log impedance has been found to be approximatelylinear with the reciprocal of these parameters. Impedance changeshave also been found to accompany endogenous and other changesin the plant. In particular, pulses of light or heat causedchanges of impedance which reflect a change of internal waterrelations and possibly transport. Suitable light and potassiumfertilizer treatments at temperatures around 4 °C enhancedthe impedance of alfalfa and its apparent resistance to frost.     

Photosystem II and oxygen regulation in Sesbania rostrata stem nodules

The tropical wetland legume Sesbania rostrata Brem. produces nitrogen-fixing stem nodules which are green and contain chlorophyll, the chloroplasts being concentrated in a hand in the inner and mid-cortex close to the nitrogen-fixing cells. The photosystem II thylakoid membrane proteins D₁, D₂ and PsbO, which are essential for photo-synthetic O₂ evolution, were shown by immunoblotting to be present in extracts of leaves and stem nodules. Immunogold labelling confirmed their presence on stem nodule thylakoids and showed that labelling was most intense in well-developed chloroplasts in the mid-cortex and least intense in the smaller, less-abundant chloroplasts adjacent to the nitrogen-fixing cells. Concentrations of the oxygen-carrying protein leghaemoglobin (Lb) did not differ between stem and S. rostrata root nodules, and Lb was localized in bacteroid-containing cells, including those immediately adjacent to the cortex, in both nodule types. Moreover, nitrogenase component 2 was localized in bacteroids within the outermost layers of infected cells, suggesting that a low pO₂ was maintained, despite the nearby chloroplasts. Nodule extracts examined by ELISA and immunoblots, using the monoclonal antibody MAC265, showed greatly enhanced expression of a 139 kDa glycoprotein in stem compared to root nodules. Immunogold labelling showed that material containing the MAC265 antigen occluded intercellular spaces, and was present in cell walls, throughout the cortex of stem nodules (particularly in the chloroplasl-rich inner and mid-cortex), but was considerably less evident in root nodules.     

Specific Labelling of Virus Proteins by Quantitative Reaction of Cysteine Residues with <Superscript>35</Superscript>S-Sulphite

POLYPEPTIDES from different sources can be compared conveniently by digesting them with proteolytic enzymes and fingerprinting the resulting smaller peptides. If peptides with identical electrophoretic and chromatographic properties are obtained, the implication is very strong that the sequences of the original polypeptides were, at least in part, the same. The need for such comparisons arises in studies of in vitro polypeptides synthesized in coupled systems directed by viral DNAs. The material synthesized in vitro must be compared with authentic virus-coded material to verify that the system is transcribing DNA to RNA and translating RNA to protein with fidelity. For viruses such as SV40 and polyoma, which can be grown in tissue culture, the virus particles grown in the presence of ³⁵S-methionine are a convenient source of virus-coded proteins. Proteolytic digests of these particles can be compared with digests of ³⁵S-methionine labelled material synthesized in vitro. Preliminary results have shown that, in the case of polyoma virus, matching peptides are obtained from virus particles and polypeptides synthesized in vitro¹.