Alan B. Bennett - US grants

A number of plant pathogens produce pectolytic enzymes whose activity degrades polygalacturonic acid within the host cell wall during the process of infection. This pectolytic cell wall degradation may contribute to pathogenesis by allowing physical penetration of the invading pathogen and perhaps in providing monomeric sugars to support energy metabolism of the pathogen. Pectic fragments produced through the action of pathogen polygalacturonases may, in addition, elicit defense responses in the infected host. Plant-derived inhibitors of pathogen polygalacturonases have been identified in several plant tissues and their presence has been shown to be associated with resistance to pathogen attack. It has been proposed that the presence of these proteinaceous polygalacturonase inhibitors may directly inhibit pathogen colonization and indirectly function to enhance the host defense response. This research will develop the biochemical and molecular genetic basis for critically assessing the role of plant inhibitors of pathogen polygalacturonases in altering pathogen susceptibility in transgenic plants. The specific objectives are to: 1) purify polygalacturonase inhibitors from pear and tomato fruit, 2) raise antibodies to purified polygalacturonase inhibitors and determine N-terminal and internal amino acid sequences, 3) construct appropriate cDNA libraries from pear and tomato and use antibody and oligonucleotide probes to isolate cDNA clones encoding pear and tomato polygalacturonase inhibitors and, 4) characterize the polygalacturonase inhibitor cDNAs by sequence analysis and patterns of mRNA accumulation. The results of these studies will provide the basis for designing strategies of over-expression or anti-sense depression of the polygalacturonase inhibitor cDNAs in transgenic plants. Losses of agricultural products to plant pathogens are substantial. While losses can often be controlled through the application of fungicides, the enhancement of endogenous plant defense systems by molecular genetic means has provided the hope of reducing the dependence upon chemical control of pathogens. The possible role of plant-produced polygalacturonase inhibitors (PGIs) in pathogen defense is widely recognized but has received little attention in recent years. This research will examine the role of PGIs in pathogen resistance. If proven effective in enhancing plant pathogen resistance, molecular manipulation will provide a powerful tool for achieving pathogen resistant crops.

This award provides funds for the establishment of a Research Training Group in Plant Cell Biology. The faculty group is a mixture of outstanding senior and junior investigators who come from a variety of disciplinary backgrounds, but share a common interest in cell biology. The funds will provide stipends for graduate students, will support research participation by undergraduate students, will defray part of the cost of the trainees' research and will enable the trainees to attend scientific meetings. In addition, funds will be used to purchase specialized research equipment to be used by trainees, and to bring investigators from other research and academic institutions to the Davis campus for seminars and symposia. In recent years, the use of new techniques for microscopy and the use of genetics and biochemistry has led to significant new knowledge of the architecture and function of the cell. Much of this progress has come through study of microbial and animal cells. The growth of knowledge of such features particular to plant cells has lagged. There is now a significant need for the training of new researchers skilled in the study of cell biology of plants. This award to one of the country's most outstanding groups of plant cell biologists should help to address the need.

This award will facilitate collaboration under the U.S.-Japan Cooperative Science Program on a project concerned with the role of ethylene as a plant hormone which regulates many aspects of plant growth and development, including fruit ripening, leaf senescence, and flower fading. The chemical precursor of ethylene in plants is the compound 1- aminocyclopropane-1-carboxylic acid (ACC). This compound in turn is produced from S-adenosylmethionine (SAM). The rate- limiting step in ethylene biosynthesis in plants has been shown to be the conversion of SAM to ACC, catalyzed by the enzyme known as ACC synthase. It is the study of this enzyme, isolated from plant tissues, that forms the subject of this cooperative project. It is known that various developmental, hormonal, and environmental factors, such as auxin treatment or plant wounding, cause the induction of ACC synthase, resulting in marked increase in ethylene production. However, the ACC synthase induced in these different ways appears to show differences in its immunological and physicochemical properties. The goal of this research is to elucidate, at both the protein and gene levels, the comparative biochemistry of ACC synthase induced in different tissues by different factors. The U.S. principal investigators in this project are Dr. Shang Fa Yang and Dr. Alan B. Bennett, Department of Vegetable Crops, University of California, Davis. Their Japanese colleagues are Dr. Hidemasa Imaseki, Nagoya University, and Dr. Shigeru Satoh, Tohoku University. The U.S. group is working with tomato and apple, and the Japanese group is studying the same process in squash and other plants. Purified materials isolated by the two groups will be exchanged and tested, and information and expertise will be shared. Results obtained during these studies should lead to further understanding of those basic plant processes regulated by ethylene. This knowledge can potentially be exploited in the control of these processes via biotechnology, with resulting significance in agriculture.

In the prior NSF grant period this laboratory cloned and characterized a higher plant (tomato) calcium-ATPase gene closely related to the sarcoplasmic/endoplasmic reticulum calcium-ATPase of animal cells. They also observed that its messenger RNA abundance increased dramatically following salt exposure, suggesting that this calcium-ATPase may play a role in the mitigation of deleterious effects of elevated cytosolic calcium, proposed to result from extracellular salt exposure. Molecular cloning of this calcium-ATPase now provides the basis to characterize it at the molecular level, including a critical assessment of its role in cellular responses to salt, and perhaps other stresses. The objectives of the current phase of this research are to: 1) establish the precise subcellular localization of this class of calcium-ATPase, 2) elucidate the basis for the accumulation of multiple calcium-ATPase messenger RNA transcripts, 3) determine the factors regulating calcium-ATPase gene expression, especially in response to stress, and 3) critically assess its physiological function in responses to environmental stress using transgenic plants with altered calcium-ATPase gene expression. %%% The regulation of cytosolic calcium levels is widely recognized as a central element of cellular regulatory processes in eukaryotes, and is essential to the proposed role for calcium as a second messenger. In addition, it has been proposed that salt and chilling stress in plants may induce, as the primary cellular insult, increases in cytosolic calcium levels. Both calcium- ATPases and a calcium/proton pumps maintain calcium homeostasis in the cytosol of plant cells. Based on extensive characterization of ATP-dependent calcium transport in membrane vesicles prepared from plant cells, there appear to be at least two calcium-ATPases with properties likely to be important in the maintenance of very low cytosolic calcium levels: one located in the plasma membrane and one in the endoplasmic reticulum membrane. In spite of the relatively extensive characterization of calcium transport activities catalyzed by plant calcium-ATPases, little success has been reported in their purification and molecular characterization. Research from this laboratory has begun to fill in these gaps, and the proposed continuation of these studies promises to provide more critical information on the cellular mechanisms involved in plant responses to their environment.

We will acquire and operate instruments for a high-throughput automated plant molecular genetics facility. The facility will comprise of an automated DNA sequencer, a DNA extraction robot, a laboratory workstation, a thermocycler, a phosphoimager, and supporting computers. The facility will primarily provide three areas of research support: DNA sequencing, quantitative analysis of gene expression, and genetic analysis. The facility will impact at least 24 productive research programs in nine departments that study plant molecular genetics, ranging from fundamental molecular genetics on model species to applied crop improvement programs. We are currently severely limited by the lack of this instrumentation. All these studies would benefit from this facility. Several experiments are impossible without it. Reliable generation of accessible DNA sequence data is a fundamental component for many biological investigations, particularly gene identification and characterization. Accurate measurement of gene expression is critical to studies on gene function. Molecular markers have now been developed for most of the species under study; these should now be utilized. This facility will allow a scale of experimentation that would be otherwise impossible in an academic setting. Automated DNA extraction as well as template preparation and analysis will allow large numbers of samples to be run with accuracy. Both the UC Davis Office of Research and the College of Agriculture and Environmental Sciences are committed to developing this facility as demonstrated by their level of financial support. Their contribution, together with that from Keygene, constitutes a 50% match to the amount requested from NSF. The College will provide technical support for a total of five years, two years beyond the funding period. We will continue to develop and apply efficient molecular marker technologies. In particular, we are collaborating with Applied Biosystems to analyze Ampli fied Fragment Length Polymorphisms as codominant markers on their instruments. This will increase the utility of their machines and allow hundreds of markers to be analyzed on many individuals. It will also reduce the generation of radioactive waste.