Beth C. Mullin

Detection of heavy metal ions using protein-functionalized microcantilever sensors

Microcantilevers functionalized with metal-binding protein, AgNt84-6, are demonstrated to be sensors for the detection of heavy metal ions like Hg(2+) and Zn(2+). AgNt84-6, a protein that has the ability to bind multiple atoms of Ni(2+), Zn(2+), Co(2+), Cu(2+), Cd(2+) and Hg(2+) was attached to the gold-coated side of silicon nitride cantilevers via linker groups. Upon exposure to 0.1 mM HgCl(2) and 0.1 mM ZnCl(2) solutions, the microcantilevers underwent bending corresponding to an expanding gold side. Exposure to a 0.1 mM solution of MnCl(2) solution did not result in a similar bending indicating a weak or no interaction of Mn(2+) ions with the AgNt84-6 protein. The microcantilever bending data were consistent with data from electrophoresis carried out on SDS-PAGE gels containing metal ions that showed protein interaction with Zn(2+) ions but not with Mn(2+) ions. Thus, we demonstrate that microcantilever bending can be used to discriminate between metal ions that bind and do not bind to AgNt84-6 protein in real time.

Phylogenetic Affinities of Datiscaceae Based on an Analysis of Nucleotide Sequences from the Plastid rbcL Gene

Nucleotide sequence data from the large subunit of plastid ribulose-1,5-bisphosphate carboxylase/oxygenase gene (rbcL) were used to infer the phylogenetic affinities of Datiscaceae relative to other dicotyledonous angiosperms and to assess whether the family is monophyletic. New rbcL sequence data from members of Datiscaceae and Cucurbitaceae were generated and analyzed with other previously available rbcL sequences. Phylogenetic reconstructions based on parsimony place Datisca closer to representatives of Cucurbitaceae and Begoniaceae than to the other two genera within Datiscaceae. Our results do not support the monophyly of Datiscaceae and suggest that a previous separation of the group at the family level may be warranted. The relationships of the Datiscaceae-Curcurbitaceae-Begoniaceae clade to other groups included in the analysis are unresolved, but analyses with smaller data sets suggest affinities with higher Hamamelidae, among which are several actinorhizal groups. Actinorhizal plants, as a group, may thus be more closely related than has been indicated by recent classification schemes.

DIVERSITY OF FRANKIA NODULE ENDOPHYTES OF THE ACTINORHIZAL SHRUB CEANOTHUS AS ASSESSED BY RFLP PATTERNS FROM SINGLE NODULE LOBES

Root nodules of the actinorhizal shrub Ceanothus were collected from seven sites from its native range. DNA was extracted from individual nodule lobes using a cetyl trimethyl ammonium bromide (CTAB) extraction procedure. Three DNA probes were used in combination with two restriction endonucleases to evaluate the extent of restriction fragment length polymorphism (RFLP) diversity within and among populations of Ceanothus endophytes. We observed some diversity using a nif DH gene probe; however there was no correlation of RFLP pattern and geographic site. Using two random Frankia probes, we observed more diversity among the Ceanothus endophytes than with the nif probe. Differences in RFLP patterns were observed among plants at a single geographic site and between geographical sites. The results demonstrated that considerable diversity exists among Frankia strains symbiotic with Ceanothus, as has been shown for pure-cultured Frankia strains isolated from other actinorhizal genera. We have also demonstrated the usefulness of this method for the study of Frankia ecology in planta.

Metallohistins: A New Class of Plant Metal-Binding Proteins

Two small multimeric histidine-rich proteins, AgNt84 and Ag164, encoded by two nodule-specific cDNAs isolated from nodule cDNA libraries of the actinorhizal host plant Alnus glutinosa, represent a new class of plant metal binding proteins. This paper reports the characterization of the purified in vitro-expressed proteins by size exclusion chromatography, circular dichroism, equilibrium dialysis, metal affinity chromatography coupled with mass spectrometry, and nuclear magnetic resonance spectroscopy. These analyses reveal that each polypeptide is capable of binding multiple atoms of Zn2+, Ni2+, Co2+, Cu2+, Cd2+ and Hg2+. A reversible shift in histidine Cε1 and Cδ2 protons in NMR analysis occurred during titration of this protein with ZnCl2 strongly suggesting that histidine residues are responsible for metal binding. AgNt84 and Ag164 are not related to metal binding metallothioneins and phytochelatins and represent a new class of plant metal binding proteins that we propose to call metallohistins. Possible biological roles in symbioses for AgNt84 and Ag164, and their potential for use in bioremediation are discussed.

Molecular analysis of actinorhizal symbiotic systems: Progress to date

The application of molecular tools to questions related to the genetics, ecology and evolution of actinorhizal symbiotic systems has been especially fruitful during the past two years. Host plant phylogenies based on molecular data have revealed markedly different relationships among host plants than have previously been suspected and have contributed to the development of new hypotheses on the origin and evolution of actinorhizal symbiotic systems. Molecular analyses of host plant gene expression in developing nodules have confirmed the occurrence of nodulin proteins and in situ hybridization techniques have been successfully adapted to permit the study of the spatial and temporal patterns of gene expression within actinorhizal nodules. The use of heterologous probes in combination with nucleotide sequence analysis have allowed a number of nif genes to be mapped on the Frankia chromosome which will ultimately contribute to the development of hypotheses related to nif gene regulation in Frankia. The use of both 16S and 23S rDNA nucleotide sequences has allowed the construction of phylogenetic trees that can be tested for congruence with symbiotic characters. In addition the development of Frankia-specific gene probes and amplification primers have contributed to studies on the genetic diversity and distribution of Frankia in the soil.

Restriction pattern analysis of genomic DNA of Frankia isolates

SummaryTotal genomic DNAs ofFrankia isolates were subjected to restriction enzyme digestion and subsequent agarose gel electrophoresis. Restriction fragment banding patterns were unique for each isolate and may therefore be used as a method to distinguish between isolates which may be morphologically indistinguishable. This method might be useful for practical purposes such as tracing specificFrankia strains during field studies.

Physical characterization of mitochondrial DNA from cotton

Mitochondrial DNA from Gossypium hirsutum was isolated from seedlings and purified. The median thermal dissociation temperature (T m ) of cotton mtDNA was determined by the thermal dissociation method of Marmur. The genome size was accurately determined by reassociation kinetic analysis using solution hybridization followed by hydroxylapatite chromatography. Two separate C O t curves were generated for Escherichia coli reference DNA

Leghemoglobin-like sequences in the DNA of four actinorhizal plants

SummaryA cloned cDNA partial copy of a soybean leghemoglobin mRNA was used to probe genomic DNA of four species of actinorhizal plants. Southern blot hybridization revealed the presence of sequences with homology to the leghemoglobin probe in DNA from Alnus glutinosa, Casuarina glauca, Ceanothus americanus and Elaeagnus pungens. The hybridization patterns of the restriction fragments revealed some fragment size conservation between the DNA of soybean and the DNA of four actinorhizal plants which are taxonomically unrelated to soybean or to each other. The results presented here indicate that globin gene sequences are much more widely distributed in the plant kingdom than has previously been thought. Furthermore, if sequence conservation is actually as high as the restriction fragment patterns suggest, the evolution of the DNA surrounding the globin sequences has been highly constrained.

Hypotheses for the evolution of actinorhizal symbioses

Actinorhizal symbioses include symbiotic associations between actinomycetes of the genus Frankia and host plants belonging to 24 genera distributed among eight higher plant families. Although all host plant species are woody dicots, there is no close phylogenetic affinity among most host plant groups. Nor is there agreement among actinorhizal biologists as to whether host plants are likely to have evolved from a common nodulated ancestor or whether symbioses have more likely arisen independently more than once resulting in the current diversity of host plant species. Knowledge of the phylogenetic relationships among actinorhizal plants as well as comparative analyses of nodule structure and function should contribute to the formulation of working hypotheses for the evolution of actinorhizal symbioses.

Differential Gene Expression in the Development of Actinorhizal Root Nodules

Actinorhizal root nodules form as a result of the interaction of actinomycetes in the genus Frankia with the appropriate host plant roots. In the actinorhizal symbiosis with Alnus, the first easily detectable sign of interaction between the plant and bacterial partners is the extensive deformation of plant root hairs. This root hair deformation which is detectable in a matter of hours has been shown not to require direct contact between bacteria and host plant (Prin, Rougier, 1987). There appears to be an as yet unidentified diffusible factor synthesized or released by frankiae in response to exposure to host plant root exudate in a manner analogous to the rhizobia/legume signaling system. Root hair deformation is followed by penetration of the root hair by Frankia filaments and the initiation of cortical cell divisions leading to prenodule formation (Berry, Sunell, 1990). The colonization of prenodule tissue is followed by the induction of a lateral root primordium in the pericycle. As the lateral root primordium grows through the infected prenodule tissue the postmeristematic cells of the now-nodule primordium are themselves infected with the microsymbiont which rapidly proliferates within host cells and begins to fix nitrogen. The nodule meristem bifurcates giving rise to the lobed nodules characteristic of Alnus.