Mary Alice Webb

Cell-mediated crystallization of calcium oxalate in plants

Plants make crystals of calcium oxalate in an intriguing variety of defined shapes. [Figure 1][1] illustrates a commercial preparation of calcium oxalate, which consists of a mix of crystals with variable sizes and irregular shapes. In contrast, crystals synthesized by plants typically exhibit quite

Functional Characterization of HFR1, a High-Mannose N-Glycan-Specific Wheat Lectin Induced by Hessian Fly Larvae

We previously cloned and characterized a novel jacalin-like lectin gene from wheat (Triticum aestivum) plants that responds to infestation by Hessian fly (Mayetiola destructor) larvae, a major dipteran pest of this crop. The infested resistant plants accumulated higher levels of Hfr-1 (for Hessian fly-responsive gene 1) transcripts compared with uninfested or susceptible plants. Here, we characterize the soluble and active recombinant His6-HFR1 protein isolated from Escherichia coli. Functional characterization of the protein using hemagglutination assays revealed lectin activity. Glycan microarray-binding assays indicated strong affinity of His6-HFR1 to Manα1-6(Manα1-3)Man trisaccharide structures. Resistant wheat plants accumulated high levels of HFR1 at the larval feeding sites, as revealed by immunodetection, but the avirulent larvae were deterred from feeding and consumed only small amounts of the lectin. Behavioral studies revealed that avirulent Hessian fly larvae on resistant plants exhibited prolonged searching and writhing behaviors as they unsuccessfully attempted to establish feeding sites. During His6-HFR1 feeding bioassays, Drosophila melanogaster larvae experienced significant delays in growth and pupation, while percentage mortality increased with progressively higher concentrations of His6-HFR1 in the diet. Thus, HFR1 is an antinutrient to dipteran larvae and may play a significant role in deterring Hessian fly larvae from feeding on resistant wheat plants.

Twinned Raphides of Calcium Oxalate in Grape (Vitis): Implications for Crystal Stability and Function

Among the higher plants that accumulate crystalline calcium oxalate, many taxa characteristically produce raphides, or needle‐shaped crystals. Substantial evidence has accumulated indicating that raphides function in plant defenses against herbivory and that their acicular shape is a critical component in proposed mechanisms for these defenses. Previous observations have shown that raphides in members of the Vitaceae are twinned crystals. In this study, the fine structure of raphides in Vitis was examined in order to determine the nature of twinning. Rotation of isolated raphides under cross‐polarized light revealed extinction patterns that indicated that raphides are twinned along their length. In cross sections of raphides, the twin plane extends across the raphides, parallel to their surfaces. The dissolution patterns observed in etched crystals indicate that the type of twinning is rotational. Parallels in other biomineralization systems indicate that the rotational nature of the twinning may increase mechanical strength. In addition, because twinning may affect crystal growth or enhance stability and persistence of crystals, it could be an important factor in allowing plant cells to produce the raphide morphology.

Orthologs in Arabidopsis thaliana of the Hsp70 interacting protein Hip

Abstract The Hsp70-interacting protein Hip binds to the adenosine triphosphatase domain of Hsp70, stabilizing it in the adenosine 5′-diphosphate–ligated conformation and promoting binding of target polypeptides. In mammalian cells, Hip is a component of the cytoplasmic chaperone heterocomplex that regulates signal transduction via interaction with hormone receptors and protein kinases. Analysis of the complete genome sequence of the model flowering plant Arabidopsis thaliana revealed 2 genes encoding Hip orthologs. The deduced sequence of AtHip-1 consists of 441 amino acid residues and is 42% identical to human Hip. AtHip-1 contains the same functional domains characterized in mammalian Hip, including an N-terminal dimerization domain, an acidic domain, 3 tetratricopeptide repeats flanked by a highly charged region, a series of degenerate GGMP repeats, and a C-terminal region similar to the Sti1/Hop/p60 protein. The deduced amino acid sequence of AtHip-2 consists of 380 amino acid residues. AtHip-2 consists of a truncated Hip-like domain that is 46% identical to human Hip, followed by a C-terminal domain related to thioredoxin. AtHip-2 is 63% identical to another Hip-thioredoxin protein recently identified in Vitis labrusca (grape). The truncated Hip domain in AtHip-2 includes the amino terminus, the acidic domain, and tetratricopeptide repeats with flanking charged region. Analyses of expressed sequence tag databases indicate that both AtHip-1 and AtHip-2 are expressed in A thaliana and that orthologs of Hip are also expressed widely in other plants. The similarity between AtHip-1 and its mammalian orthologs is consistent with a similar role in plant cells. The sequence of AtHip-2 suggests the possibility of additional unique chaperone functions.

Purification of allantoinase from soybean seeds and production and characterization of anti-allantoinase antibodies.

Allantoinase catalyzes the hydrolysis of allantoin to allantoic acid, a reaction important in both biogenesis and degradation of ureides. Ureide production in cotyledons of germinating soybean (Glycine max L.) seeds has not been studied extensively but may be important in mobilizing nitrogen reserves. Allantoinase was purified approximately 2500-fold from a crude extract of soybean seeds by differential centrifugation, heat treatment, ammonium sulfate fractionation, ethanol fractionation, and fast protein liquid chromatography (Pharmacia) with Mono-Q and Superose columns. The purified enzyme had a subunit size of 30 kD. Polyclonal antibodies produced against the purified protein titrated allantoinase activity in a crude extract of seed proteins. Antibodies recognized the 30-kD band in western blot analysis of crude seed extracts, indicating that they were specific for allantoinase.

Soybean Root Nodule cDNA Encoding Glutathione Reductase

GSH is widely distributed in organisms and has diverse functions in protein synthesis, sulfur storage, and protection against a variety of stresses (Meister and Anderson, 1983). GR (EC 1.6.4.2) is a flavoprotein that catalyzes the reduction of GSSG to reduced GSH using NADPH as the reducing cofactor. It is a key enzyme in the GSH-ascorbate cycle, which provides protection against oxidative stress, particularly in photosynthetic tissues of plants (Foyer and Halliwell, 1976). Tobacco transformed with the gor gene, encoding GR in Escherichia coli, showed mixed results in resistance to oxidative stress (Aono et al., 1991; Foyer et al., 1991). Recently, a cDNA clone encoding GR was isolated from a cDNA library of pea leaves (Creissen et al., 1991). An N-terminal leader sequence encoded by the pea cDNA was consistent with targeting of the gene product to the chloroplast. In root nodules, enzymes of the GSH-ascorbate cycle have been suggested to function in peroxide scavenging and protection against other oxidative stresses (Dalton et al., 1986). In soybean, GR activity was much higher in nodules than in uninfected roots and was enhanced in nodules exposed to higher than normal p 0 2 (Dalton et al., 1991). GR activity also was about 4-fold higher in effective nodules than in ineffective nodules, with a range of factors controlling effectiveness (Dalton et al., 1993). Soybean (Glycine max) nodules contain both homo-GSH as well as GSH, and the combined concentration of thiol tripeptides in nodules was calculated to be about 1 m~ (Dalton et al., 1993). Recent studies examining the role of GSH in relation to pathogen defense responses (Wingate et al., 1988; Edwards et al., 1991) have implications for GSH function during nodule development as well. A full-length cDNA encoding soybean nodule GR (Table I) was isolated serendipitously in screening a soybean nodule cDNA library with antibodies against nodule allantoinase. The deduced amino acid sequence of the cDNA was found to share a high degree of homology to sequences for GR in other organisms and was 90% similar and 83% identical to pea GR (Creissen et al., 1991). The soybean GR sequence also had an N-terminal leader sequence characteristic of a chloroplast transit peptide, which would target the gene product to nonphotosynthetic plastids in root nodules. South’

Immunoaffinity purification and comparison of allantoinases from soybean root nodules and cotyledons.

Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyzes the conversion of allantoin to allantoic acid in the final step of ureide biogenesis. We have purified allantoinase more than 4000-fold by immunoaffinity chromatography from root nodules and cotyledons of soybean (Glycine max [L.] Merr.). We characterized and compared properties of the enzyme from the two sources. Seed and nodule allantoinases had 80% identity in the first 24 amino acid residues of the N terminus. Two-dimensional gel electrophoresis of the purified enzymes showed that multiple forms were present in each. Allantoinases from nodules and cotyledons had very low affinity for allantoin with a Km for allantoin of 17.3 mM in cotyledons and 24.4 mM in nodules. Both had activity in a broad range of pH values from 6.5 to 7.5. In addition, purified allantoinase from both sources was very heat stable. Enzyme activity was stable after 1 h at 70[deg]C, decreased gradually with heating to 85[deg]C, and was lost at 90 to 95[deg]C. Although these studies have revealed some differences between allantoinases in seeds and nodules, the differences were not reflected in key enzyme properties. The immunoaffinity approach enabled purification of allantoinase from soybean root nodules and simplified its purification from cotyledons, thereby allowing characterization and comparison of the enzyme from the two sources.

Calcium Oxalate Accumulation in Malpighian Tubules of Silkworm (Bombyx mori)

Silkworm provides an ideal model system for study of calcium oxalate crystallization in kidney‐like organs, called Malpighian tubules. During their growth and development, silkworm larvae accumulate massive amounts of calcium oxalate crystals in their Malpighian tubules with no apparent harm to the organism. This manuscript reports studies of crystal structure in the tubules along with analyses identifying molecular constituents of tubule exudate.