Phosphomannose isomerase in Nicotiana tabacum L. NT1 and Apium graveolens var. dulce L. cell suspension cultures

by 1978- Barb, Adam Wesley

BARB, ADAM WESLEY. Phosphomannose isomerase in Nicotiana tabacum L. NT1 and Apium graveolens var. dulce L. cell suspension cultures. (Under the direction of Drs. D.M. Pharr and J.D. Williamson) Phosphomannose isomerase (PMI) [E.C. 5.3.1.8] is a key enzyme required for Man metabolism in plants, and PMI activity is important in both mannitol metabolizing and mannitol non-metabolizing plants. In this manuscript, two studies are presented; one that describes PMI activity in a mannitol non-metabolizing plant, Nicotiana tabacum L. NT1 cell suspension cultures, and one that describes the partial purification of PMI from a mannitol metabolizing plant, Apium graveolens var. dulce. Wild type cultures of NT1 cells grew slowly with Man as the sole carbohydrate source, doubling every 158 h, whereas NT1 cells doubled every 26 h when grown on Glc as the sole carbohydrate source. A mutant line of NT1 cells was selected from the wild type culture by repeated subculture into fresh Man medium, and after three months, exhibited a doubling time of 38 h on Man as the sole carbohydrate source. Additionally, this mutant culture exhibited a five-fold higher PMI activity level than the wild type culture from which it was selected. Interestingly, we noted that this mutant culture had a decreased growth rate on Glc, with a culture doubling time of 31 hours, and was found to have more than a 50% reduction in hexokinase (HK) [2.7.1.1] activity. In the second study, A. graveolens cell suspension cultures were shown to have high levels of PMI activity (200 µmol hr-1 g-1 fresh weight) and provided an excellent starting material for the purification of PMI. PMI from Glc-grown A. graveolens cultures was purified 23.5-fold with a 6% total recovery of PMI activity, using ammonium sulfate precipitation, gel filtration chromatography, and ion exchange chromatography. Low recovery of PMI activity was presumably because this activity was not stable. Although protease inhibitors in the extraction buffer resulted in the recovery of nearly twice the amount of nonspecific protein and PMI activity, neither protease inhibitors nor the addition of zinc to the enzyme solution aided in the maintenance or recovery of PMI activity in purification steps past ammonium sulfate precipitation. Additionally, different affinity chromatography resins did not bind PMI activity, but might still serve as a useful tool later in the purification of the enzyme. Phosphomannose isomerase in Nicotiana tabacum L. NT1 and Apium graveolens var. dulce L. cell suspension cultures by Adam Wesley Barb A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Master of Science Department of Horticultural Science and Plant Physiology Program Raleigh 2002 Approved by Biography Adam Wesley Barb was born in Milwaukee, Wisconsin on August 14, 1978 to Stuart Clair and Paula Dye Barb. Adam’s university education began in the Fall of 1996 at Purdue University (West Lafayette, Indiana), and he received a Bachelor of Science degree in Horticulture Science in May of 2000. While an undergraduate student at Purdue, Adam was persuaded to participate in research by Dr. Robert Joly. As a student, Adam took part in research to find and characterize T-DNA insert mutants in Arabidopsis thaliana plants that rendered the plants more sensitive to salinity and cold stress. While working closely with Dr. Hisashi Koiwa under the auspices of Dr. Ray Bressan and Dr. Mike Hasegawa, Adam decided to further his education and pursue a graduate degree in plant science. In August of 2000, Adam began his education as Master of Science student at the Department of Horticultural Science and as a member of the Plant Physiology Program at North Carolina State University. ii