The biosynthesis of (1,3)-B-linked glucan in higher plants is catalyzed by the plasma membrane-bound enzyme callose synthase (UDP-Glc: (1,3)-B-glucan synthase). Callose synthase activity is ubiquitous in isolated membrane fractions from a wide variety of plants, and has been hypothesized to be a deregulated form of cellulose synthase. Therefore, to understand the biochemical basis for the regulation of cell wall polysaccharide biogenesis and wound healing, it is necesary to identify and characterize the polypeptide subunits of polysaccharide synthases such as calloses synthase, and the genes which encode them. Although callose synthase has proven to be a stubborn enzyme to purify to homogeneity by standard protein fractionation techniques, rapid progress has been made towards identifying the polypeptide components of callose synthases from various sources. Comparisons of polypeptide profiles between our system, Beta vulgaris L., and other plants show the emergence of common polypeptide profiles. During the previous two years, through photoaffinity labeling, improved enrichment procedures and most recently polypeptide depletion, we have identified UDP-Glc-binding subunits of 57-,31- and 29-kD, as well as a group of instrinsic hydrophobic polypeptides of 92, 83, 43 and 27 kD. Our working hypothesis, upon which the proposed experiments are predicated, is that the one or more of these polypeptidesare part of a multi-subunit callose synthase complex. This project will proceed along two lines. The first will be a continuation of our efforts to unambiguously identify callose synthase subunits, and will consist of polypeptide-depletion experiments, topographic analysis using vesicles of defined sidedness, photolabeling studies using azido-probes and limited proteolysis. We will continue to raise antibodies against individual enzyme subunits. Second, we plan to clone and sequence a structural gene(s) encoding subunit shown to be required for enzyme activity. Identification and cloning of callose synthase subunits should pave the way for resolving the longstanding question of whether (1,3) and (1,4) B-linked glucans are biosynthesized by a single enzyme or distinct enzymes where each produces a distinct linkage type. The availability of amino acid sequences and cDNA for callose synthase subunits will reveal whether sequence homologies exist with known bacterial cellulose synthase genes, and therefore define the extent to which callose and cellulose synthases are structurally related. This research will also help pave the way to study gene expression during cell wall biogenesis. %%% Cellulose (1,4-B-D-glucan) is the world's most abundant macromolecule, with an estimated 1011 tons biosynthesized per year. It is a major component of plant cell walls providing structural integrity and protection against invading plant pathogens. Callose, the (1,3)-B-linked counterpart of cellulose, is physiologically important since its synthesis is induced in response to wounding and infection. Callose is also found in plant structures such as pollen tubes, sieve plants and in the cell walls of some monocots. B-glucans from plants and yeast are are economically important due to their unique agrilcultural, physiological and nutritional properties. Recent studies show that B-Glucans of mixed linkage ((1,3),(1,4)-B-glucan) from oats and (1,3)-B-glucan from yeast have the ability to lower cholesterol in hypercholesteremic individuals when significant levels are incorporated into the diet. The pharmaceutical industry is interested in fungal glucan synthases because they represent potential molecular targets for new drugs which are needed to treat systemic fungal infections. Very little is known about the biochemical mechanism by which glucans are produced. This project focuses on expanding our knowledge of the structure and function of (1,3)-B-glucan synthase, commonly known as callose synthase, a cell wall biosynthetic enzyme complex ubiquitous in higher plants. In the near-term, one objective is continued application of basic protein chemistry to make further progress towards the unambiguous identification of the protein components of CS. In addition, our knowledge of the CS complex has reached the point where a molecular biology component is warranted. Thus, a second objective is to clone selected components of the CS complex in order to obtain deduced amino acid sequences for CS polypeptides. We will then determine whether similarities exist with known sequences of cellulose synthase from cellulose-producing microorganisms. This would indicate whether cellulose and CSs are structurally related. This research will also help to gain an improved understanding of callose deposition as part of the wound response. Research on the biosynthesis of plant-derived biopolymers has important scientific and economic implications. It is critical for U.S.-based labs to continue to play a significant role in these efforts.
