Progress 10/01/10 to 09/30/15

Outputs
Target Audience:Plant scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A protein inside banana raphides was identified. In vitro experiments showed that a portion of the protein sequence promoted growth of calcium oxalate crystals with an acicular morphology.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Xiuli Li, Wenjun Zhang, Jianwei Lu, Lixue Huang, Defeng Nan, Mary Alice Webb, Francois Hillion, Lijun Wang. 2014. Templated biomineralization on selfassembled protein nanofibers buried in calcium oxalate raphides of Musa spp. Chem. Mater. 26 (12) 3862-3869.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Scientists interested in roles of biomineralization and mechanisms that control synthesis of intracellular minerals in biological organisms Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Results have been disseminated to the research community via publication in a scientific journal. What do you plan to do during the next reporting period to accomplish the goals? Our research will determine whether raphides present in other plants also contain protein that templates their growth and whether the protein is the same or different from that discovered in banana raphides. In addition, the characteristics and potential roles of other proteins associated with raphides in plants will continue to be explored.

Impacts
What was accomplished under these goals? Many plants synthesize organized bundles containing a hundred or more needle-shaped crystals, called raphides. These crystals, typically composed of calcium oxalate, have been shown to function in discouraging herbivory, thereby minimizing damage to plants. Banana plants contain raphides in abundance throughout their leaves, as do many other plants. In this study we have discovered that in banana leaves a protein nanofiber is present inside each individual raphide, and further, that the nanofiber functions in templating the growth and development of raphides into their characteristic needle shape. In addition, a number of other proteins associated with banana raphides were identified and sequenced, and these proteins may have other important roles in raphide synthesis and assembly.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Xiuli Li, Wenjun Zhang, Jianwei Lu, Lixue Huang, Defeng Nan, Mary Alice Webb, Francois Hillion, Lijun Wang. 2014. Templated biomineralization on selfassembled protein nanofibers buried in calcium oxalate raphides of Musa spp. Chem. Mater. 26 (12) 3862-3869.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Colleagues in plant biology Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A postdoctoral researcher working with the Chinese collaborators was trained by Dr. Webb in essential methods used to accomplish this project. How have the results been disseminated to communities of interest? A publication on this research has been drafted. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The elongated "needle" shape of raphides is critical to their function in defense against herbivores. Raphides are synthesized in highly specialized cells in plants, and they typically are produced in bundles consisting of several hundred raphide crystals. Our research has focused on learning what organelles and macromolecules within these specialized cells are important in raphide synthesis. Previous studies in our lab have shown that an organic matrix associated with developing raphides within the cells that produce them is important in initiating and shaping raphides. Objective 1: In collaboration with a research group in China led by a crystallographer, a protein that accumulates within the raphides of banana has been discovered and characterized. A postdoctoral researcher in the Chinese group was trained by Dr. Webb in essential methods used to accomplish this project. Objectives 2 and 3: Sequence was obtained from the protein of interest, and a peptide, a portion of the protein, was synthetically produced. When calcium oxalate crystals were grown outside the plant in solutions containing the peptide, crystals were longer than those grown in solutions without the peptide added. This preliminary evidence indicates that the peptide could influence raphide shape in vivo, enhancing the length of crystals as they are synthesized within cells. Crystal length is an important factor in the needle-like shape of raphides, contributing to their defensive function in plants. Information from these results could be applied to other plants to improve their ability to discourage herbivory. A publication based on this research has been drafted.

Publications

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: Raphides are needle-shaped crystals composed of calcium oxalate that occur in many angiosperms. Previous research supports the function of raphides in plant defense against herbivores. We have performed a survey of angiosperm phylogeny to determine the distribution of raphides among phylogenetic groups. Results of our survey revealed gaps separating groups with raphides, indicating that raphides might have evolved independently multiple times during the evolution of angiosperms. In an initial survey we observed that raphides in three plant orders deviated from the generic morphology characteristic of most raphides, which comprise straight, smooth needle-shaped crystals that taper to points at each end. Plants in the Alismatales typically have raphides with grooved sides, barbed edges, and spear-shaped ends. Raphides in members of the Vitales consist of twinned crystals with the twin plane along the length of the raphides. In the order Caryophyllales, raphides were observed to have a unique morphology consisting of elongated triangle-shaped facets. In continuing research we have expanded the breadth of our investigations by examining families and genera within each of these orders to determine whether the morphologies previously observed are consistent. Raphide morphology does appear to be consistent within the Alismatales and the Vitales, but in the Caryophyllales, the unusual triangular raphides we observed occur in only one family, the Aizoaceae. A poster focused on this research was presented at the Gordon Conference on Biomineralization in August 2012. PARTICIPANTS: Mary Alice Webb was principal investigator. Michael Zanis contributed knowledge about angiosperm phylogeny. TARGET AUDIENCES: Target audiences are scientists involved in research on biomineralization and biomaterials. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our investigations have provided new information about raphide microstructure in relation to angiosperm phylogeny that serves as a context for other researchers who study mineral structures composed of calcium oxalate, calcium carbonate, and silica in plants. Our observations also provide a basis for examining structure/function relationships. Because plants cannot run away from insects and other herbivores, they must rely on a variety of inherent defenses against these threats. Specific features of raphides, such as their morphology or associated macromolecules, could be important to their effectiveness in defending plants against herbivory. In previous research on raphides in the Vitales, we hypothesized that twinning would increase the strength of the raphides. We have recently agreed to collaborate with an engineering group to test that hypothesis, specifically, to determine the mechanical strength of the twinned raphides in the Vitales and to compare them to raphides in other taxa. Because raphides are synthesized within specialized cells in plants, investigations of raphides also are relevant in the broad context of biomineralization to studies of mineralized structures such as bones, teeth, and protective shells in animals. Relating microstructural features of raphides to their mechanical strength might also provide inspiration for applications in the fields of biomaterials and biomimetics.

Publications

• No publications reported this period

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Dr. Webb has studied raphides, needle-shaped crystals of calcium oxalate, shown to be important in plant defense against herbivores. Reviews of published literature, as well as information on the Missouri Botanical Garden site on Angiosperm Phylogeny, were used to survey phylogenetic groups to determine which orders of plants produce raphides. In collaboration with Dr. Michael Zanis, we noted that the distribution of raphides across the angiosperms appeared to indicate that this specialized crystal morphology may have evolved independently multiple times during the evolution of angiosperms. We found that raphides are not present in basal angiosperms, although some magnolids have raphide-like crystals. In contrast, raphides are very common in monocots, occuring in most orders. Within eudicots raphides are absent in basal eudicots but present in selected core eudicot lineages. Raphides occur in the Vitales, which are considered sister to the rosids, but were apparently lost during radiation of the rosid I lineage. The occurrence of raphides is scattered within the rosid II lineage, in groups sister to the asterids and in the asteroid I lineage. We used light microscopy with crossed polars and scanning electron microscopy to examine the microstructure of raphides and raphide-like crystals in selected angiosperms. Dr. Webb was invited to present results of this study at the 11th International Symposium on Biomineralization, held in Noosa, Australia, in a talk entitled "Evolution of calcium oxalate raphides in angiosperms (flowering plants)." PARTICIPANTS: Dr. Mary Alice Webb, Dr. Michael Zanis TARGET AUDIENCES: Plant biologists interested in calcium nutrition and calcium oxalate crystallization; animal scientists interested in biomineralization PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Examination of raphides and raphide-forming cells revealed that they have unique features in different phylogenetic groups. In most plants that produce raphides, the needle-shaped crystals occur in organized bundles, and all raphides in a bundle are similar in length. However, raphide-like crystals in basal angiosperms were not organized in bundles and raphides within a single cell varied in length. In the monocot order Alismatales, raphides have unique fine structure with grooves along the length of the raphides and spear-like end structures, as well as specialized cells that eject raphides. In all other monocot orders observed raphides exhibited facets along the length of the raphide that terminated in pointed ends. Raphides in members of the Vitales (eudicot, sister to rosids) are uniquely twinned along their length with opposing pointed and forked end structures (Webb and Arnott, 2001). In the Caryophyllales (sister to asterids) we found that raphides have an unusual asymmetric structure with thick and thin facets forming a triangular shape in a face view of thick facets and a narrow rectangular profile on thin facets. Our studies have also shown that organization of raphides within bundles and the composition of mucilage associated with raphide bundles differ among groups. Many previously published studies of raphide development indicate that cell structure, as well as conformation of membrane structures enclosing raphide synthesis also vary among different phylogenetic groups. Results of our studies and consideration of previous studies are consistent with the idea that raphides have evolved independently multiple times during the evolution of angiosperms.

Publications

• No publications reported this period

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Dr. Webb mentored a visiting graduate student, WenJun Zhang, from Huazhong Agricultural University in Wuhan, China. The student was trained in methods for analyzing proteins, including SDS-gel electrophoresis for separating proteins according to molecular weight, and a variety of methods for staining proteins in the gel. She also learned methods used for viewing plant samples at high resolution in the scanning electron microscope. Preliminary results from WenJun's research were presented at the Midwest American Society of Plant Biologist meeting in Spring 2010l. PARTICIPANTS: Dr. Mary Alice Webb led the project as principal investigator. Ms. WenJun Zhang, visiting graduate student, conducted laboratory experiments under Dr. Webb's guidance. TARGET AUDIENCES: Plant scientists interested in calcium oxalate PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We began developing Musa acuminata as a model system for studying calcium oxalate crystallization in plants. In banana we examined the structure and distribution of cells that form bundles of needle-shaped crystals, called raphides, within the petiole. We applied methods developed in our lab to isolate raphide bundles from the petioles. From isolated bundles, we extracted proteins for analysis by SDS-polyacryamide gel electrophoresis. Results showed that an assortment of proteins with a wide range in molecular mass was associated with raphides. Profiles of raphide-associated proteins from banana were very similar to those previously observed in grape, but not identical. We determined that everal proteins in banana stained with PAS (periodic acid/Schiff's reagent), which identifies glycoproteins. In anticipation of proteomics analysis, we tested methods for zinc staining of SDS-gels. In collaboration with Dr. Nicholas Carpita, we also analyzed carbohydrates associated with raphide bundles in banana. Results were consistent with the presence of mostly glucomannans, distinct from raphide-associated carbohydrates previously identified in grape. These studies provide a foundation for analysis of protein sequences and their identification using proteomics methodology.

Publications

• No publications reported this period

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Most flowering plants, or angiosperms, produce crystalline calcium oxalate, which typically accumulates in the vacuoles of specialized cells. These cells provide high-capacity sinks for calcium storage and sequestration in a highly insoluble and metabolically inactive form. In selected plant families calcium oxalate accumulates in the form of needle-shaped crystals, called raphides. Raphides have important functions in plant defense against herbivory, and toxic or acrid macromolecules associated with raphides may affect the palatability of plants that contain them. We have recently conducted studies using high resolution scanning electron microscopy to compare raphide microstructure in evolutionarily divergent plant families. These ultrastructural studies will provide a platform for comparative biochemical studies and for examining evolutionary pressures on raphide form and function. Raphides occur in the majority of monocot orders, but not in the Poales (grasses). They also occur in comparatively few orders among eudicots. In all cases raphides form bundles composed of hundreds to thousands of crystals; however, substantial variation was observed among angiosperm taxa in the fine structure of raphides comprising the bundles. In the monocot order Alismatales, plants notorious for causing toxic reactions in herbivores have raphide structure that appears to enhance delivery of raphide-associated toxins. For example, in some groups raphides contain grooves extending along their length, and some also have flared serrated ends resembling arrowheads. Other representatives of the order have raphides with barbed or serrated edges. Raphides in eudicots are not commonly associated with toxicity, and they lack characteristic ultrastructural features of defensive raphides in monocots. In representatives of the order Caryophyllales, raphides are long and thin with pointed ends, but no other distinguishing fine structure. In representative plants of the Vitales each raphide consists of two crystals, twinned along the length of the raphide, with one crystal of the twin rotated in relation to the other. The rotational relationship between crystals in the twins, like layers of plywood, may enhance raphide strength and stability. Continuing studies will provide additional data about raphide microstructure in relation to angiosperm phylogeny. These studies will be conducted in concert with biochemical studies, which previously have allowed us to identify putative raphide-associated proteins in grape. Comparative biochemical studies will reveal whether proteins associated with raphides in different plant taxa are similar or divergent. These studies may also allow us to separate proteins essential to raphide development from others that function primarily in plant defense to reduce herbivory. PARTICIPANTS: During this project period research was conducted by the principal investigator, Mary Alice Webb. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Needle-shaped crystals composed of calcium oxalate, called raphides, constitute one element in an arsenal of defensive compounds that plants produce to protect themselves. Raphides have been demonstrated to be important in deterring herbivores, animals that feed on plants. Basic research is needed to understand how raphides and chemical compounds associated with them have evolved in concert to enhance their defensive function. The comparative studies in progress will provide new information that could be applied in protection of crop plants against insects and other herbivores.

Publications

• No publications reported this period

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Many plants make biominerals composed of calcium oxalate and/or calcium carbonate to regulate calcium, as well as to enhance plant defenses against herbivores. The extent to which plants partition calcium into these biominerals, as well as their form and distribution, can affect factors such as insect resistance and availability of calcium in foods. Our recent research has focused on 1) the form and distribution of calcium oxalate deposits examined in an evolutionary context, 2) cellular factors that control accumulation of calcium oxalate in grape, and 3) calcium mineralization of defensive trichomes in the plant family Brassicaceae. In surveying the morphology of calcium oxalate crystals in angiosperms, we have observed and cataloged the diversity in their form and distribution in order to develop hypotheses about their evolution in plants. Using proteomics methodology we have identified proteins associated with crystals in grape, and have provided evidence that membranes enclosing crystals are related to the tonoplast. We have shown that trichomes in Arabidopsis and other members of the Brassicaceae have considerable mineral content, tentatively identified as amorphous calcium carbonate. We are examining how trichome mineralization may be affected in genetic mutants of Arabidopsis. PARTICIPANTS: Mary Alice Webb, Aaron Wyman (postdoctoral research associate) TARGET AUDIENCES: Fellow scientists in plant biology, biomineralization, and materials engineering PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Research has added to knowledge about the scope and form of calcium oxalate accumulation in plants relevant to understanding the evolution of calcium oxalate mineralization. Additional knowledge has been gained about protein constituents of cellular membranes that compartmentalize calcium oxalate in plants, and this will contribute to greater understanding about how cells control the synthesis of these deposits. Studies of calcium carbonate mineralization in Arabidopsis trichomes provide the basis for using this model system to study genetic factors influencing trichome mineralization and the effects of trichome mineralization on plant defense against herbivory.

Publications

• No publications reported this period

Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: We are examining how grape plants control synthesis of calcium oxalate to produce needle-shaped crystals, called raphides. In previous studies, we identified numerous raphide-associated proteins using proteomics approaches. These included ion transporters, a variety of chaperones, enzymes, and cytoskeletal elements. One transporter identified was a member of the Slc26a family of anion transporters, which has been shown in humans to transport oxalate. Using RT-PCR, we have cloned several cDNAs from grape that encode members of this family. One of these has significant similarity to a human oxalate transporter. In addition, we have cloned a cDNA from grape encoding a variant of a raphide-associated protein, called Hip (Hsp70-interacting protein). Whereas a previously cloned cDNA encoded an unusual chimeric protein consisting of Hip joined to a carboxy thioredoxin domain, the Hip variant recently cloned has no thiorecoxin and exhibits substantial sequence similarity with human Hip. We are investigating whether grape Hips and interacting chaperones might regulate calcium transport in membrane chambers enclosing developing raphides. In collaborative research, we are examining effects of substituting each of the plant Hips for human Hip in a yeast model system to elucidate their function. Continuation of studies examining calcium-binding properties of Hip has demonstrated that an acidic domain within the protein is both necessary and sufficient for calcium binding. Additional proteomics analyses have emphasized comparative studies relating proteins associated with raphides to proteins associated with calcium oxalate crystals in kidney-like organs of silkworm. Many of the same proteins extracted from grape raphides have also been found in protein extracts of silkworm crystals. These comparative studies will yield information about which proteins are critical to crystal formation. In other research we have used nanoSEM to examine early stages of raphide development in situ in grape roots. Successive stages in development of raphides and the specialized cells that form them can be observed in differentiating root tips, which exhibit files of raphide-forming cells. Previous studies with TEM had revealed individual raphides initiating in small vesicles, proliferating in provacuoles, and eventually forming an organized bundle in mature vacuoles of the crystal-forming cells. Observations with nanoSEM indicated that vesicles containing developing raphides formed an interconnected network within the cytoplasm. Intravesicular compartments were frequently observed, and raphides developing together were commonly interconnected by fibrillar material of unknown composition. We plan to examine whether fibrils connecting the raphides are composed of cytoskeletal elements, which could play an important role in controlling the organization of raphides within the vacuole. Results of these studies were presented at an International Conference on Urolithiasis, an annual meeting of the American Society of Plant Biologists, and an International Conference on the Chemistry and Biology of Mineralized Tissues. PARTICIPANTS: Mary Alice Webb Aaron Wyman, Postdoctoral Research Associate TARGET AUDIENCES: plant biologists scientists studying biomineralization

Impacts
Calcium oxalate crystals in plants provide several important functions, including regulation of excess calcium and protection against herbivory. Partitioning of calcium into crystalline form is an important factor in calcium regulation at the cell and tissue levels. Needle-shaped crystals of calcium oxalate in plants play important roles in chemical defenses against herbivores by carrying bitter or toxic proteins through the skin of herbivores. To understand how plant cells control the crystallization process, we have focused on identifying and characterizing proteins associated with calcium oxalate crystals in plants. Information gained about how these crystal-associated proteins function will advance our understanding about how cells control calcium partitioning into calcium oxalate. Because calcium oxalate accumulates inside specialized plant cells, the plant system also provides a model for understanding cellular factors that influence mineral deposits, such as bones and teeth, in a variety of organisms. In addition to its relevance to plant growth, development, and survival, knowledge about calcium oxalate crystallization in plants has a wide variety of potential applications, including development of low-oxalate foods, identification of drugs for treatment of calcium oxalate kidney stones, and biomimetic synthesis of materials.

Publications

• Wyman, A.J. and M.A. Webb. 2007. Calcium oxalate accumulation in Malpighian tubules of silkworm (Bombyx mori), pp. 407-411. In: Renal Stone Disease: First International Urolithiasis Research Symposium. A.P. Evan, J.E. Lingeman, and J.C. Williams, eds. American Institute of Physics, Melville, NY.

Progress 10/01/05 to 09/30/06

Outputs
Needle-shaped crystals of calcium oxalate, called raphides, form uniquely in plants and function in plant defense against predators. We have been studying how grape plants control the synthesis of calcium oxalate to produce raphides. Raphides develop in intravacuolar membrane systems, called crystal chambers. We have initiated proteomics approaches using MALDI-TOF mass spectrometry to identify raphide-associated proteins with potential functional roles in crystallization. This approach has provided identification of a number of very interesting candidates in grape for further study. These are listed below along with explanations of their potential roles in crystal formation: 1) High affinity sulfate transporter 1 belongs to the Slc26a family of putative sulfate transporters. Recent studies in other laboratories showed that these transporters mediate chloride-oxalate exchange in mice and demonstrated further that Slc26a6 null mice not only exhibited highly increased excretion of urinary oxalate in comparison to wild type, but also developed kidney stones. This protein represents an excellent candidate for an oxalate transporter in the grape crystal cells. We will initiate its study in grape by examining localization of expression via in situ hybridization. 2) Annexin IV/annexin p33 can form calcium channels in a membrane or modulate other calcium channels. Calcium transport into the compartments formed by crystal chambers is critical to calcium oxalate formation. A similar annexin occurs in matrix vesicle membranes that function in the initiation of crystallization in bone, which is composed of calcium phosphate. 3) Calgranulin B is an S100 calcium binding protein previously identified only in vertebrates. Previous research has shown that it inhibits growth of calcium oxalate crystals in vitro, and has been found in human kidney stones composed of calcium oxalate. We have not identified any sequences corresponding to calgranulin in the Vitis EST databases, and it is possible that this protein is a contaminant. Before proceeding with further study, we will evaluate by RT-PCR whether it is expressed in grape. If so, calgranulin could function as an inhibitor of crystal growth essential to synthesis of the raphide morphology. Calgranulins typically interact with annexins, therefore, interaction of calgranulin with the annexin described above will be examined in future research. 4) Carbonic anhydrase II is commonly associated with a variety of calcification systems in animals. Absence or inhibition of this enzyme in humans can disrupt normal calcium regulation with serious consequences including osteoporosis, kidney stone formation, and cerebral calcification. It is not immediately obvious how this protein might function in raphide formation; however, its role in calcification processes in humans and other biomineralization systems clearly warrant further examination of the protein in grape. Further studies of these proteins and the genes encoding them in grape will reveal new information about how plant cells synthesize raphides.

Impacts
Calcium oxalate crystals in plants provide several important functions, including regulation of excess calcium and protection against herbivory. Because calcium oxalate accumulates inside specialized plant cells, the plant system also provides a model for understanding cellular factors that influence mineral deposition in a variety of organisms. To understand how plant cells control the crystallization process, we have focused on identifying and characterizing proteins associated with calcium oxalate crystals in plants. Information gained about the functions of these crystal-associated proteins will advance our understanding about how cells control calcium partitioning into calcium oxalate. In addition to its relevance to plant growth and development, knowledge about calcium oxalate crystallization in plants has a wide variety of potential applications, including development of low-oxalate foods, identification of drugs for treatment of calcium oxalate kidney stones, and biomimetic synthesis of materials.

Publications

• No publications reported this period

Progress 10/01/04 to 09/30/05

Outputs
We previously identified the chaperone Hip (Heat shock protein 70-interacting protein) in a screen for proteins associated with calcium oxalate crystals in grape. In animals Hip is involved in the regulation of steroid hormones. The function of Hip is not known in plants. Hip includes a number of functional domains, most of which have been characterized. However, the function of an acidic domain near the N-terminus of the protein has not been determined. We have examined the function of the acidic domain in grape, mammalian, and two Arabidopsis Hips. All of these Hips contain a prominent acidic domain, but the amino acid sequence varies within the domain among the different Hips. We have expressed the plant Hips in E. coli and assayed them for calcium binding using calcium ligand blotting. In all cases, full-length recombinant protein binds calcium, whereas truncated protein without the acidic domain does not bind calcium. In addition, we found that rat Hip also bound calcium. We have now expressed the acidic domain from grape Hip and are testing whether that alone is sufficient to bind calcium. We have also initiated studies to examine Hip interacting proteins and to test whether Hip interactions are regulated by calcium. Using anti-Hsp70 antibodies to immunoprecipitate Hsp70-interacting proteins from Arabidopsis extracts, we have identified a number of interacting proteins, many of unknown function. Using anti-Hip antibodies to immunoprecipitate interacting proteins, we have also identified several Hip-interacting proteins. Sequencing of these proteins is in progress to determine their identification. We plan to do these immunoprecipitation experiments with plants grown in media with differing calcium concentrations, as well as plants subjected to selected other stresses. To determine how altered expression of Hip affects plant growth and development, we have obtained homozygous mutants in one of two genes in Arabidopsis. On defined media mutants grew more slowly than wild type and produced fewer siliques. We plan to examine growth and development of mutants in comparison to wild type on media with differing calcium concentrations and a variety of stresses. We have also initiated a project to identify additional proteins associated with calcium oxalate crystals in grape. We isolated crystals from grape leaves and extracted proteins associated with these crystals. Proteins were separated by SDS-PAGE, and selected polypeptides were extracted from the gel and digested with trypsin. Digested samples were submitted for sequence analysis by MALDI-TOF mass spectrometry, and analysis of sequence data is in progress.

Impacts
Calcium oxalate crystals in plants provide several important functions, including regulation of excess calcium and protection against herbivory. Because calcium oxalate accumulates inside specialized plant cells, the plant system also provides a model for understanding cellular factors that influence mineral deposition in a variety of organisms. To understand how plant cells control the crystallization process, we have focused on identifying and characterizing proteins associated with calcium oxalate crystals in plants. Information gained about the functions of these crystal-associated proteins will advance our understanding about how cells control calcium partitioning into calcium oxalate. In addition to its relevance to plant growth and development, knowledge about calcium oxalate crystallization in plants has a wide variety of potential applications, including development of low-oxalate foods, identification of drugs for treatment of calcium oxalate kidney stones, and biomimetic synthesis of materials.

Publications

• No publications reported this period

Progress 10/01/03 to 09/29/04

Outputs
In our research we are examining cellular factors influencing the accumulation of calcium oxalate crystals in plants. In grape plants calcium oxalate develops in the vacuoles of specialized cells. Previous research has shown that these cells function in sequestering excess calcium within plant tissues. We are currently studying a cochaperone named Hip to determine whether it plays a role in detecting elevated calcium and initiating a signaling cascade that leads to cell differentiation. We previously showed that Hip binds calcium, and we hypothesize that Hip interaction with another chaperone, Hsp70, may be regulated by calcium. We are developing a system to examine Hip/Hsp70 interaction in vitro so that we can determine what external factors might regulate the interaction. In addition, we are examining Arabidopsis mutants resulting from T-DNA inserts in a gene encoding Hip. Homozygous recessive mutants have no obvious morphological phenotype under normal growth conditions. In further study, we will examine the internal anatomy of mutant plants. We also have designed experiments to evaluate whether their response to a variety of stresses, including elevated calcium supply, differs in comparison to wild type plants. The Arabidopsis genome contains two genes encoding different forms of Hip. By performing RT-PCR with gene-specific primers we have examined expression of each gene in Arabidopsis leaves, stems, roots, flowers, and siliques and found that both genes were expressed in all parts of the plant. These results do not rule out the possibility of differential regulation at the level of protein translation or activation within organs or tissues, and we plan to examine that next. In a related project we are developing and applying a novel method to identify proteins within a complex mixture that are functionally active in modifying crystal growth. We are using this method to analyze proteins extracted from plant crystals. In grape we have identified specific proteins that inhibit crystal growth, as well as others that promote crystal growth and/or aggregation. We have also assayed proteins extracted from calcium oxalate crystals in water hyacinth, which produces the same morphological type of crystal as grape. We have now identified crystal-growth-modifying proteins in water hyacinth that have similar size and functional characteristics as those previously identified in grape. We are now extending our studies to plants with different morphological types of crystals to determine whether they have different functionally active proteins. Proteins identified in this research have potential for widespread applications in medicine and materials science.

Impacts
We study proteins and other molecules that influence the growth of crystals and the cells that form crystals inside plants. Plant crystals contain calcium, and they are important in protecting plant cells from excess calcium in their environment, as well as in discouraging pests that may feed on plants. We are studying how a specific protein previously identified in our research is regulated by calcium. We think that this protein may function in a pathway that initiates crystal development within plant cells. In addition, we have developed a new method that allows us to analyze proteins extracted from plant crystals and to identify those that function in promoting or inhibiting crystal growth. Proteins identified by this method could be used in improving plants via transgenic technology. They also could have applications in medicine, such as artificial bone implants, or in materials science, where they might allow synthesis of crystals with particular sizes and shapes useful in technology.

Publications

• No publications reported this period

Progress 10/01/02 to 09/30/03

Outputs
Many plants make crystals of calcium oxalate. A primary function of these crystals is to regulate excess calcium within plants. In some plants crystals develop in a needle-shaped morphology, termed raphide, that deters herbivory. Using grape plants as a model system we are identifying cellular factors that may influence the development of the raphide shape. Previously, we used an immunological approach to identify raphide-associated proteins (RAPs) and to clone genes encoding these proteins. Recent research has focused on functional analysis of one of these RAPs. This protein is a chimera consisting of two domains, one similar to human Hip (Hsp70-interacting protein) and another similar to thioredoxin (Trx). Its function has not been examined in plants. We expressed the full-length grape Hip/Trx protein in bacteria and showed that the recombinant protein binds calcium. In contrast, a truncated recombinant protein from which the putative calcium-binding motif was deleted does not bind calcium. These results indicate that the Hip/Trx protein could be regulated by calcium. We hypothesize that it functions as a sensor for calcium stress within the crystal-forming cells and may initiate the developmental pathway that leads to calcium oxalate accumulation. To gain additional information about the function of the Hip/Trx protein, we have identified null mutants in Arabidopsis in the gene encoding Hip. Work is also in progress to identify null mutants in the Arabidopsis gene encoding a Hip/Trx protein similar to the one present in grape. We will examine the phenotype of these mutants under normal growth conditions and under calcium stress. In other research we have examined the effects of proteins extracted from grape raphides on the growth of calcium oxalate in vitro. We have developed a novel method that allows us to identify specific proteins within a complex mixture that promote or inhibit crystal growth in vitro. We are presently working on refining this method using proteins extracted from grape raphides. We expect that the method will also be useful in identifying proteins that are functionally active as crystal growth modifiers in other mineralizing tissues in plants and animals.

Impacts
We are studying proteins that potentially control growth of crystals in plants. These crystals are important in regulating excess calcium within plants, as well as in discouraging pests that may feed on plants. We have shown that a specific protein previously identified in our research is regulated by calcium. This supports the idea that this protein may function in a pathway that initiates crystal development within plant cells. In addition, we have developed a new method that allows us to analyze a complex mix of proteins and to identify proteins within the mixture that function in promoting or inhibiting crystal growth. This method should be applicable to other mineralizing tissues, such as developing bones and teeth in animals, as well as in plants.

Publications

• Stitsworth, A. and M.A. Webb. 2003. Effects of matrix components from crystal cells in grape on growth of calcium oxalate crystals in vitro. Conn Tiss Res 44 (Suppl.1): 338.
• Klanrit, P., J. Cavaletto, W. McDowell, G. Thomspon, M.A. Webb. 2003. Characterization of cDNAs putatively encoding raphide-associated proteins in grape. Conn Tiss Res 44 (Suppl. 1): 362.

Progress 10/01/01 to 09/30/02

Outputs
We are studying the accumulation of crystalline calcium oxalate in grape. Specialized cells in grape plants produce bundles of calcium oxalate crystals with a needle-shaped morphology, and these crystals may be important in calcium regulation and in plant defense against herbivory. We are trying to understand how plant cells control growth and development of the crystals to produce their specialized shape. We have cloned several genes that putatively encode proteins associated with the crystals and may represent proteinaceous components of membranes that compartmentalize the crystals. Our current focus is on a chimeric protein with domains similar to human Hip (Hsp70-interacting protein) and thioredoxin. The role of this protein in plants is unknown. Using antibodies against human Hip, we have shown that a Hip-like protein is present in extracts of proteins from isolated grape crystals. We have shown further that Hsp70, the interacting protein for Hip, is present in the same extracts. The deduced amino acid sequence for Hip contains a putative calcium-binding motif, and we have begun to test the protein directly to determine whether it binds calcium. Preliminary studies indicate that the recombinant protein expressed in E. coli does bind calcium. Future studies will examine whether the protein is regulated by calcium. Immunolocalization studies to pinpoint the intracellular location of Hip within crystal-forming cells in grape roots are in progress. We have also initiated a project to examine oxalate accumulation in soybean seeds. We have examined the structure and distribution of calcium oxalate crystals in the seed and have found that crystals are abundant and evenly distributed throughout the cotyledons. Future studies will examine the effects of environmental, physiological and cellular factors on oxalate production in soybean.

Impacts
Calcium oxalate crystals play important roles in plant growth and development, including disposal of excess calcium and protection against pests. Our research will contribute to understanding how plants shape crystals and place them in the appropriate place within the plant according to their function. Although crystals may be beneficial to plants, they can be harmful to humans and other animals that consume plants as food. Our research will potentially lead to the ability to modify crystal formation in plants, e.g., to increase crystal accumulation for plant protection and other benefits, or alternatively, to block crystal formation in plant foods.

Publications

• No publications reported this period

Progress 10/01/00 to 09/30/01

Outputs
Our research has examined accumulation of calcium oxalate crystals in the vacuoles of specialized cells in grape. Previous research has shown that these cells function in sequestering excess calcium within plant tissues. In grape we previously isolated several cDNAs that putatively encode protein constituents of crystal chambers, membranes that compartmentalize developing calcium oxalate crystals within the vacuole. We are studying these cDNAs to understand the role of the gene products in the crystallization process. Recent research has focused on a cDNA encoding a chimeric protein with both Hip (Hsc-70 interacting protein) and thioredoxin domains. In mammals Hip is a regulatory protein that modulates the activity of chaperone heterocomplexes, but its role in plants is unknown. Sequence analysis revealed a putative calcium-binding motif in the predicted protein encoded by the grape cDNA. We have shown that antibodies against human Hip recognize a protein associated with calcium oxalate crystals in grape. We have also expressed the grape cDNA in E. coli, and recombinant protein will be used for antibody production, as well as tests to determine whether the protein binds calcium. Studies in the near future will examine localization of the protein in developing crystal-forming cells of grape. Sequence analysis showed that the Arabidopsis genome encodes two forms of Hip, one similar to mammalian Hip and a second similar to the Hip/thioredoxin chimera from grape. This will allow genetic approaches using Arabidopsis to examine Hip function in plants. Additional studies in grape have examined a cDNA encoding a kinesin-related protein. Antibodies against a peptide sequence conserved in kinesins recognized a protein associated with calcium oxalate crystals in grape. The kinesin may be involved in movement and organization of crystals within developing crystal cells. Research is in progress to characterize this cDNA and to express it in bacteria for further study of the kinesin-like protein encoded by the cDNA.

Impacts
Calcium oxalate accumulation in plants provides a model for studies of cellular factors affecting biomineralization. Our research is examining proteins associated with calcium oxalate crystals in grape and the genes that encode these proteins. Our research will lead to understanding how the plant cell controls the crystallization process. This knowledge has a wide variety of potential applications outside of plant growth and development, including materials science and nanotechnology, bone development and fabrication in animals, and human nutrition.

Publications

• Webb MA, JM Cavaletto, P Klanrit, G Thompson. 2001. Orthologs in Arabidopsis thaliana of the Hsp70 interacting protein Hip. Cell Stress and Chaperones 6 (3), 247-255.
• Stitsworth A, MA Webb. 2001. Effects of matrix components from crystal cells in grape on growth of calcium oxalate crystals in vitro. Proceedings, 7th International Conference on The Chemistry and Biology of Mineralized Tissues, Nov 2001 (abstract).
• Klanrit P, J Cavaletto, W McDowell, G Thompson, MA Webb. 2001. Characterization of cDNAs putatively encoding raphide-associated proteins in grape. Proceedings, 7th International Conference on The Chemistry and Biology of Mineralized Tissues, Nov 2001 (abstract).

Progress 10/01/99 to 09/30/00

Outputs
We are studying development of calcium oxalate crystals in the vacuoles of specialized cells in grape. Previous research indicated that these cells function in calcium regulation by sequestering excess calcium. In grape we have isolated several cDNAs that putatively encode protein constituents of membranes that compartmentalize developing calcium oxalate crystals within the vacuole. We are characterizing these cDNAs to understand the role of the gene products in the crystallization process. One of the cDNAs encodes a protein with multiple functional domains, suggesting roles in calcium binding, chaperone interaction, and redox regulation. Another appears to encode a kinesin-like protein that may be involved in membrane or vesicle trafficking in the crystal-forming cells. We have also initiated studies to identify mutants in calcium oxalate formation in Medicago truncatula.

Impacts
Our studies are aimed towards understanding the role of calcium oxalate crystallization in plant growth and development and enhancing basic knowledge about how plant cells and organelles mediate this process.

Publications

• Arnott, H. J. and M. A. Webb. 2000. Twinned raphides of calcium oxalate in grape (Vitis): implications for crystal stability and function. Int. J. Plant Sci. 161:133-142.

Progress 10/01/98 to 09/30/99

Outputs
We are studying accumulation of crystalline calcium oxalate in the vacuoles of specialized cells in grape. Previous research indicated that these cells function in calcium regulation by sequestering excess calcium. In grape we have isolated several cDNAs that putatively encode protein constituents of membranes that compartmentalize developing calcium oxalate crystals within the vacuole. We have confirmed that the cDNAs represent transcripts expressed in developing grape leaves. One of these cDNAs exhibits no significant similarity to known sequences. Another appears to encode a kinesin-like protein and a third a protein that interacts with heat shock chaperonin. In order to study the gene products we have expressed portions of the cDNAs in bacteria to produce recombinant protein. We have also initiated studies to identify mutants in calcium oxalate formation in Medicago truncatula. Our studies are aimed toward understanding the role of calcium oxalate crystallization and enhancing basic knowledge about how plant cells and organelles mediate this process.

Impacts
We are presently examining and characterizing the organic matrix associated with developing crystals in grape (Vitis), and we have isolated cDNA clones Our research will help us understand how and why plants produce calcium oxalate crystals of specific sizes and shapes.

Publications

• Webb, M.A. 1999. Cell-mediated crystallization of calcium oxalate in plants. Plant Cell 11: 751-761

Progress 10/01/97 to 09/30/98

Outputs
We are studying accumulation of calcium and crystallization of calcium oxalate in the vacuoles of specialized cells in grape and tobacco. Previous research indicated that these cells function in calcium regulation by sequestering excess calcium. In grape we are characterizing four cDNAs that putatively encode protein constituents of intravacuolar membranes that compartmentalize calcium oxalate crystals. We have confirmed that the cDNAs represent transcripts expressed in developing grape leaves. The putative translation product of one cDNA has similarity to both heat-shock interacting protein and thioredoxin in separate domains. Two other cDNAs exhibit no significant similarity to known sequences. We are currently examining localization of expression within grape tissues. In tobacco we used mRNA differential display to identify nine cDNAs that putatively represent differential expression in seedlings with and without calcium oxalate accumulation. Our studies are aimed towards enhancing basic knowledge about how plant cells and organelles mediate crystallization of calcium oxalate.

Impacts
(N/A)

Publications

• Cavaletto, J. 1997. Raphide-associated proteins in grape (Vitis labrusca). M.S., Purdue University, 48 pp.
• Racicot, V.M. 1998. Calcium accumulation and calcium oxalate formation in tobacco (Nicotiana tabacum L.) M.S., Purdue University, 60 pp. Eccleston, V., J. Cavaletto, W. McDowell, M. A. Webb. 1997. Differentiation of cells specialized for calcium oxalate crystallization in grape. Plant Physiol. 114:313. (abstract)
• Racicot, V.M., C.M. Rinderle, K.G. Ragothama, M.A. Webb. 1997. Effects of nutrient supply and light upon deposition of calcium oxalate in tobacco leaf. Plant Physiol. 114:313. (abstract)
• Webb, M.A., R. Burnett, J. Cavaletto, V. Eccleston, W. McDowell, V. Racicot, C. Rinderle. 1998. Cell-mediated crystallization of calcium oxalate in grape and tobacco. Proceedings, Keystone Symposium on Plant Cell Biology. (abstract)

Progress 10/01/96 to 09/30/97

Outputs
In grape and tobacco we are studying cells specialized to accumulate crystalline calcium oxalate within their vacuoles. Previous research indicates that these cells function in calcium regulation by sequestering excess calcium. In grape we have isolated four cDNAs that putatively encode protein constituents of membranes that compartmentalize crystal formation in the vacuole. Characterization of these clones is in progress. Ultrastructural studies of developing crystal cells in grape roots revealed that crystals initiate in cytoplasmic vesicles prior to incorporation into the vacuole. In physiological studies of tobacco we have found that calcium concentration in the external medium and light intensity both have significant effects on the induction of crystal-forming cells in the leaf. In addition, we have developed tobacco cell cultures that accumulate crystalline calcium oxalate in response to elevated calcium supply. These will be used in future research to identify genes expressed during calcium oxalate accumulation. These studies will lead to enhanced understanding about how plant cells mediate the crystallization process to sequester calcium oxalate.

Impacts
(N/A)

Publications

• Racicot VR, Rinderle CR, Raghothama KG, Webb MA., 1997. Effects of nutrient supply and light upon deposition of calcium oxalate in tobacco leaf. Plant Physiol. 114: 313.
• Eccleston V, Cavaletto J, McDowell W, Webb MA., 1997. Differentiation of cells specialized for calcium oxalate crystallization in grape. Plant Physiol. 114: 313.

Progress 10/01/95 to 09/30/96

Outputs
In our research on crystallization of calcium oxalate in plant cells, we have continued to study the intravacuolar membrane systems, termed crystal chambers, that differentiate within crystal-forming cells of grape. We have constructed cDNA expression libraries from mRNA isolated from grape leaves at two different stages of development. Both libraries have been screened with antibodies that recognize components of the crystal chambers, and numerous potential clones have been isolated for the genes encoding chamber proteins. In other studies we have examined differentiation of cells that form calcium oxalate in tobacco. We have also defined growth conditions under which tobacco does not produce calium oxalate. We are currently using differential screening methods to isolate genes differentially expressed during calcium oxalate crystallization in tobacco. In other research we have shown that tobacco seedlings grown in strontium accumulate greater than 1% of their dry weight in strontium and incorporate strontium into calcium oxalate crystals.

Impacts
(N/A)

Publications

• CAVALETTO, J., V. ECCLESTON, AND M. A. WEBB. 1996. Molecular and biochemical analysis of proteins associated with calcium oxalate crystals in grape. Plant Physiol. 111:145.
• RACICOT, V.M., C. M. RINDERLE, AND M.A. WEBB. 1996. Development of.

Progress 10/01/94 to 09/30/95

Outputs
In our research on calcium oxalate crystallization in plants, we have continued to examine the intravacuolar membrane systems, termed crystal chambers, that differentiate within crystal-forming cells of Vitis labrusca (grape). We have used immunoaffinity chromatography to isolate a 60-kD polypeptide that is present in the crystal chambers. N-terminal sequencing of this polypeptide was unsuccessful, because it was blocked at the amino terminus. Recent efforts have been directed to obtaining sufficient quantities of the polypeptide for enzymatic digestion to produce peptides from which we can get internal sequence information. We have also worked out an appropriate digestion protocol by doing trial digestions with BSA, which is approximately the same size as our protein of interest. In addition, several protocols for mRNA isolation from developing grape leaves were evaluated, and the best of these was used to isolate mRNA for construction of a cDNA library. We initiated antibody screening of the cDNA library to isolate a clone for the gene encoding the 60-kD immunoreactive polypeptide but have not yet isolated a clone. In other studies we have examined calcium tolerance in crystal-forming plant species, as well as other species that do not form crystals. Other research has examined calcium oxalate formation in a cell culture system.

Impacts
(N/A)

Publications

• WEBB, M. A., J. CAVALETTO, AND J. BELL. 1995. Ontogeny and composition of membranes involved in initiation and development of calcium oxalate crystals in Vitis (grape). J. Cell. Biochem. Suppl. 19A: 154. (Abstract)
• WEBB, M. A., J. M. CAVALETTO, N. C. CARPITA, L. LOPEZ, AND H. J. ARNOTT. 1995. The intravacuolar organic matrix associated with calcium oxalate crystals in leaves of Vitis. Plant J. 7: 633-648 and cover.

Progress 10/01/93 to 09/30/94

Outputs
Studies of nitrogen metabolism focused on allantoinase in ureide biogenesis in soybean. Allantoinase was isolated from soybean seeds and root nodules by immunoaffinity chromatography, and the heat stability of the purified enzyme from the two sources was examined. Allantoinase from seeds and nodules retained activity after heating for one hour at 70(degree)C. Enzyme activity decreased gradually with heating to 85(degree)C and was lost at 90-95(degree)C. These studies of purified allantoinase indicate that heat stability is intrinsic to the protein and not conferred by extrinsic factors in the crude extract. Research in calcium metabolism focused on the organic matrix associated with calcium oxalate crystallization in Vitis labrusca. We are presently examining the ontogeny and composition of crystal chambers, membranes that enclose developing crystals of calcium oxalate in the vacuole. Polyclonal antibodies produced against the crystal chambers recognize polypeptides of 60 and 70 kDa in Western blot analysis. These antibodies have been used to purify the immunoreactive polypeptides by immunoaffinity chromatography, and the polypeptides will be characterized. Antibodies against the crystal chamber proteins are also being used to screen a cDNA expression library from grape leaves. Additional immunological studies have shown that a vacuolar type ATPase is prominent in the crystal chambers, suggesting that they are developmentally related to the tonoplast.

Impacts
(N/A)

Publications

• BELL, J. A., and M. A. WEBB. 1994. Comparison of seed and nodules allantoinase purified by antibody affinity chromatography. Plant Physiol. 105:158.
• WEBB, M. A., and J. CAVALETTO. 1994. Intravacuolar membrane systems associated with calcium oxalate formation in grape (Vitis labrusca). Plant Physiol. 105:55.
• TANG, X., and M. A. WEBB. 1994. Soybean root nodule cDNA encoding glutathione reductase. Plant Physiol. 104:1081-1082.
• DAMSZ, B., J. M. DANNENHOFFER, J. A. BELL, and M. A. WEBB. 1994. Immunocytochemical localization of uricase in peroxisomes of soybean cotyledons. Plant Cell Physiol. 35: 979-982.

Progress 10/01/92 to 09/30/93

Outputs
Studies of nitrogen metabolism focused on allantoinase in ureide biogenesis in soybean. An antibody affinity column constructed with anti-allantoinase antibodies was used to isolate allantoinase from soybean seeds and root nodules. We found that the seed and nodule enzyme share 80% identity in the first 25 amino acids of the amino terminus. Both are stable to high temperature, and both have a very high K(subscript M) for allantoin. We have also isolated a full-length cDNA clone for the gene encoding glutathione reductase from a soybean nodule cDNA library. The deduced amino acid sequence had an N-terminal leader sequence characteristic of a plastid targeting sequence. Southern analysis suggested that glutathione reductase was encoded by a multi-gene family, and we have used the cDNA clone as a probe to isolate seven additional cDNA clones. Sequencing of these clones indicates that they have similar coding sequences but different 3'-untranslated regions. In situ hybridization studies showed that transcript was present in the infected region of developing root nodules and not in the root cortex. Research in calcium metabolism focused on the organic matrix associated with calcium oxalate formation in Vitis labrusca. We produced polyclonal antibodies against proteins of membrane chambers in which calcium oxalate crystals develop. The antibodies recognize two polypeptides in Western blot analysis. We have also characterized polysaccharides associated with calcium oxalate formation in Vitis.

Impacts
(N/A)

Publications

Progress 10/01/91 to 09/30/92

Outputs
Studies of nitrogen metabolism have focused on allantoinase, an enzyme involved in ureide biogenesis in soybean. We have used antibodies previously produced against allantoinase purified from soybean seeds to screen cDNA libraries from soybean nodules and seeds in unsuccessful attempts to isolate clones for allantoinase. We have also used the antibodies to prepare an antibody affinity column, which we have used for isolating and characterizing allantoinase from soybean root nodules. In other research we have shown that uricase, previously thought to be a nodule-specific protein in soybean, is present in soybean cotyledons where it is localized in glyoxysomes. We have isolated a full-length cDNA clone for the gene encoding glutathione reductase from a soybean nodule cDNA library. The deduced amino acid sequence had an N-terminal leader sequence with characteristics of a plastid targeting sequence. The clone appears to be part of a multi-gene family, and we have used the cDNA clone as a probe to isolate seven additional clones from a genomic library. Research in the area of calcium metabolism continued to focus on the organic matrix associated with calcium oxalate formation in Vitis labrusca. We are in the process of producing polyclonal antibodies against proteins of the membrane chambers in which calcium oxalate precipitates. The antibodies will be used to screen a cDNA library to isolate clones for genes encoding the membrane proteins. They will also be used in immunolocalization studies.

Impacts
(N/A)

Publications

• WEBB,M.A. AND H. ARNOTT. 1992. Isolation and characterization of the matrix associated with calcium oxalate formation in Vitis leaf idioblasts. Katherine Esau Intl. Symp.:21.
• BELL, J. AND M.A.WEBB. 1992. Purification and characterization of allantoinase from soybean root nodules buy antibody affinity chromatography. Ninth Intl. Cong. Nitrogen Fixation.
• DAMSZ,B., J.DANNENHOFFER, J.BELL, AND M.A.WEBB. 1992. Immunolocalization of uricase in soybean cotyledons and nodules using antibodies elicited against native and flutaraldehyde-fixed protein. Ninth Intl. Cong. Nitrogen Fixation.
• TANG,X. AND M.A.WEBB. 1992. Cloning and characterization of cDNAs encoding glutathione reductase from soybean root nodules. Ninth Intl. Congress Nitrogen Fixation.

Progress 10/01/90 to 09/30/91

Outputs
In studies of ureide biogenesis we have purified allantoinase from soybean seedsand produced polyclonal antibodies against the purified enzyme. Antibodies have been used to examine allantoinase expression in soybean seeds and developing seedlings in relation to allantoinase activity patterns. Western blot analysis has shown a shift in protein mobility that appears to relate to changes in activity. Antibodies have also been used to examine and characterize allantoinase in soybean root nodules. These studies have shown that different isozymes are predominant in seeds and nodules. Work is in progress to screen a soybean nodule expression library to identify potential cDNA clones for the gene encoding allantoinase. Research in the area of calcium metabolism in plants has focused on characterizing the organic matrix associated with calcium oxalate crystal formation in leaves of Vitis mustangensis and V. labrusca. Studies have shown that the matrix consists of two phases, a water-soluble outer matrix and membrane-like chambers that surround individual crystals. Analysis of protein content has revealed that an assortment of polypeptides are present in both phases of the matrix, including a prominent glycoprotein component. Carbohydrate analysis has revealed a mixture of sugars and glucuronic acid in the water-soluble phase of the matrix. X-ray microanalysis has shown that both calcium and potassium are present in the matrix.

Impacts
(N/A)

Publications

• WEBB, M. A. 1991. Analysis of the organic matrix associated with calcium oxalate crystals in Vitis mustangensis. In: Mechanisms and Phylogeny of Mineralization in Biological Systems, ed. by S. Suga and H. Nakahara, Springer-Verlag, Tokyo,.