Chemical screening of inhibitors specific for MdDOX-Co that cause an apple columnar tree-shape

MdDOX-Co, the ectopic expression of which is considered to cause the apple columnar tree shape, belongs to the 2-oxoglutarate-dependent dioxygenase (2ODD) family. It adds a hydroxyl group to position 12 of gibberellins (GAs). However, the 2ODD enzymes related to GA biosynthesis and catabolism are phylogenetically distinct from MdDOX-Co. Thus, it is possible that substrates other than GAs exist in MdDOX-Co. To identify the previously unidentified substrate(s) of MdDOX-Co, we searched for MdDOX-Co-specific inhibitors. Chemical screening using gas chromatography-mass spectrometry was performed to investigate the effects of 2400 compounds that inhibited the catalytic reaction of MdDOX-Co, but not the catabolic reaction of GA 2-oxidase, an enzyme involved in GA catabolism. By applying two positive compounds in Arabidopsis, a chemical 3-((2-chloro-6-fluorobenzyl)thio)-5,7-dimethyl-5H-pyrazolo[3,4-e][1,4,2]dithiazine-1,1-dioxide designated as TPDD that did not inhibit GA biosynthesis was selected. The structure-activity relationships among the TPDD analogs were also obtained.