MOLECULAR-CLONING OF VIOLAXANTHIN DE-EPOXIDASE FROM ROMAINE LETTUCE AND EXPRESSION IN ESCHERICHIA-COLI

Plants need to avoid or dissipate excess light energy to protect photo system II (PSII) from photoinhibitory damage. Higher plants have a con served system that dissipates excess energy as heat in the light-harve sting complexes of PSII that depends on the transthylakoid Delta pH an d violaxanthin de-epoxidase (VDE) activity, To our knowledge, we repor t the first cloning of a cDNA encoding VDE and expression of functiona l enzyme in Escherichia coli, VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized protein s. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cy steine-rich region, (ii) a lipocalin signature, and (iii) a highly cha rged region, The E. coil expressed enzyme de-epoxidizes violaxanthin s equentially to antheraxanthin and zeaxanthin, and is inhibited by dith iothreitol, similar to VDE purified from chloroplasts. This confirms t hat the cDNA encodes an authentic VDE of a higher plant and is unequiv ocal evidence that the same enzyme catalyzes the two-step mono de-epox idation reaction, The cloning of VDE opens new opportunities for exami ning the function and evolution of the xanthophyll cycle, and possibly enhancing tight-stress tolerance of plants.