John C. Watson - US grants

9877094

Abstract

This project involves the acquisition of a phosphoimager system for molecular biology research and training. The instrument will be housed in the Department of Biology at Indiana University-Purdue University at Indianapolis and will serve 5 major and two minor users in the department of Biology and two major users in the Department of Chemistry. All major and minor users employ conventional autoradiography, fluorography and/or chemiluminescent detection with film on a routine basis. However, these techniques are limited by the narrow linear response range of film and by the lengthy exposure times required for the fluorographic detection of weak energy isotopes such as 3H and 14C. Phosphoimager analysis has several key advantages over autoradiography and fluorography. First, phosphoimager screens have a linear response range of five orders of magnitude as opposed to only two for autoradiography. Second, phosphoimager exposure times are generally only one tenth those required for autoradiography and fluorography. This is especially advantageous for the detection of weak energy isotopes where fluorography exposure times of weeks or months are often required. Third, the phosphoimager data can be quantified and imported into computer drawing programs for accurate quantitative analysis. This system will complement existing molecular biology equipment in the laboratories of the major and minor users. Moreover, the high degree of accuracy and efficiency afforded by phosphoimager analysis will improve the quality and quantity of molecular biology research and teaching performed by the investigators.

Protein kinases are regulatory enzymes found in all cell types. They are involved in a vast array of responses to hormonal, nutritional, developmental, and environmental stimuli. The overall goal of the work proposed is to elucidate the roles played by selected protein kinases in the development of young seedlings. Two protein kinases from Arabidopsis named WAG1 and WAG2 are the focus of this proposal. Mutations in both WAG1 and WAG2 abolish the expression of these genes. Nevertheless, the wag1 and wag2 single mutants exhibit no detectable affect on the plant. However, when the wag single mutants are genetically crossed to generate wag1/wag2 double mutants, they do show a readily observable difference compared to their non-mutant counterparts. Seedlings of the double mutants exhibit a pronounced root growth defect when grown on vertically-oriented Petri plates: the roots possess a wavy pattern. Root waving of this type is observed in normal seedlings only when the Petri plates are leaned backward from vertical (inclined at pitches less than 90). It is proposed that WAG1 and WAG2 are negative regulators of root waving in Arabidopsis.

To elucidate the roles that WAG1 and WAG2 play in root growth, the specific aims of the proposed research are:

(1) To ask whether altering the amount or enzymatic activity of WAG1 and WAG2 affects the root waving response in transgenic Arabidopsis seedlings.

(2) To explore the underlying mechanisms for the wavy root phenotype in wag1/wag2 mutants, particularly with regard to the effect of gravity on root growth and the transport of and response to the plant growth hormone auxin.


Broader Impacts:
This project will provide training opportunities for an undergraduate student, a graduate student, and a postdoctoral associate. The two WAG genes under study encode protein kinases that when mutated confer an unusual growth pattern on seedling roots. Understanding the role of the WAG kinases will provide fundamental insight into the cellular pathways regulating root development that have not yet been defined. Root waving can be viewed as an adaptive response that may enhance the ability of a root to navigate its way around impenetrable objects encountered in the soil. As such, understanding the process of root waving and the genes that regulate it might contribute to other research areas, such as ecological physiology. Moreover, manipulating root waving genetically might improve a young seedlings ability to become established in the field, and thus provide agricultural benefits as well.