A mutant (NS 458) of Nicotiana sylvestris (Spegazzini and Comes) unable to synthesize leaf starch was isolated in the M2 generation following ethyl methanesulfonate mutagenesis by testing with iodine. Segregation ratios in reciprocal F2 progenies showed that the starchless phenotype resulted from a recessive mutation in a single nuclear gene. DEAE-agarose chromatography showed that the mutant is grossly deficient in plastid phosphoglucomutase (EC 2.1.5.1) activity. The structure of the enzyme is changed, as evidenced by increased Michaelis constants and by the prolonged activation period (>40 minutes) observed when the enzyme is assayed in triethanolamine buffer rather than imidazole buffer. The activity of the wild-type enzyme with saturating glucose 6-P alone was 7% of the activity when saturating glucose 1,6-P2 was also present. The results suggest that glucose 1,6-P2 is both an effector and a dissociable reaction intermediate. The growth rate of mutant and wild-type plants were not significantly different in continuous light and on an 8-hour dark, 16-hour light cycle and the mutants grew normally under greenhouse conditions. The mutant supports growth during diurnal periods of darkness by vacuolar storage of sugars instead of chloroplast storage of starch. The simplification in metabolism achieved by blocking the diversion of plastid fructose-6-P to starch facilitates the induction of oscillations in CO2 fixation.Carbon partitioning in photosynthetic tissues is determined by complex regulatory interactions which are only partially understood (9, 10, 17, 24). The metabolic intermediates being partitioned themselves influence the rates of CO2 fixation, hence description of the factors limiting net carbon assimilation ultimately requires modeling the system as a whole. In studying complex systems a useful method is to examine a simplified system in which one component has been eliminated or, failing that, modified in a known way. Such an approach requires the isolation ofviable mutants with defined lesions in the metabolism of photosynthetic carbon in a species convenient for both biochemical and physiological studies.A nuclear mutation reducing the activity of plastid PGI' by 50% has been recovered in Clarkia xantiana (11). The mutant had higher levels of sucrose, lower levels of starch, and reduced leaf weight relative to wild type, indicating that wild-type levels of plastid PGI are required to maintain the normal carbohydrate status and growth rate. Starchless mutants have been obtained in Arabidopsis thaliana lacking plastid PGM (1) and ADP-Glc pyrophosphorylase (12). The PGM mutant accumulated high 'Abbreviations: PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; RuBP, ribulose 1,5-bisphosphate. levels ofsoluble sugars in place ofstarch and displayed reductions in photosynthesis and growth rate relative to wild type when grown on a 12 h photoperiod. The apparent importance of starch formation in the regulation ofcarbon metabolism in Clarkia and Arabidopsis prompted us to search for starchless m...
Enzymes vary in the degree of their substrate specificity.1™4 Some are highly exacting, and others tolerate major changes in the substrate with little change in reaction rate. For a given substrate, however, the stereoselectivity with respect to the reacting centers is generally observed to be as complete as the methods of examination can reveal.5It is apparent, therefore, that the study of enzyme reaction stereochemistry differs profoundly from the study of reactions in free solution. In chemical terms, an enzyme is both a chiral reagent and a chiral solvent; the formation and breaking of bonds take place under conditions where accessibility and motion of reactants are severly constrained in ways that cannot be deduced from their chemical structures. In the context of an active site, water behaves as a stereospecific reagent and carbonium ions, carbanions, and perhaps radicals seem to possess configurational sta-This work has been supported, in part, by NSF Grant GB-42247-X, USPH Grant GM-20940, and Grants CA-06927 and RR-05539 from the Commonwealth of Pennsylvania to the Institute for Cancer Research.
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