Molecular cloning and physiological analysis of an invertase isoenzyme in Helianthus tissues

Abstract

A soluble acid invertase activity isolated from Helianthus tuberosus (Jerusalem artichoke) shoots and analyzed by immunochromatography using polyclonal yeast antibodies, represents around 5% of the total invertase activity. This invertase isoenzyme was also isolated from dormant tuber parenchyma. In these partially dormant tissues, the specific activity of this isoenzyme is low suggesting a partial inactivation of the invertase molecules. Polyacrylamide gel electrophoresis of immunopurified fractions yields similar levels of the 58 kDa polypeptide both in shoots and dormant tubers, but with much lower activity of the enzyme in the tubers. A cDNA library was constructed in pUEX 1 from poly (A)+ RNA extracted from Jerusalem artichoke tubers. This library was screened for invertase using (i) a Bacillus subtilis invertase DNA probe and (ii) anti-yeast invertase antibodies. A recombinant clone of approximately 1.8 kb size was selected by these two methods. Using Northern blots, a temporal sequence in the expression of invertase gene was observed during the breaking of dormancy with the main level after 8 weeks of cold treatment at 4 degrees C. A 2.5 kb transcript was detected, translation of which would yield a 97 kDa polypeptide representing the precursor of Jerusalem artichoke invertase.