Arabidopsis HARMLESS TO OZONE LAYER protein methylates a glucosinolate breakdown product and functions in resistance to Pseudomonas syringae pv. maculicola.

Almost all of the chlorine-containing gas emitted from natural sources is methyl chloride (CH(3)Cl), which contributes to the destruction of the stratospheric ozone layer. Tropical and subtropical plants emit substantial amounts of CH(3)Cl. A gene involved in CH(3)Cl emission from Arabidopsis was previously identified and designated HARMLESS TO OZONE LAYER (hereafter AtHOL1) based on the mutant phenotype. Our previous studies demonstrated that AtHOL1 and its homologs, AtHOL2 and AtHOL3, have S-adenosyl-l-methionine-dependent methyltransferase activities. However, the physiological functions of AtHOLs have yet to be elucidated. In the present study, our comparative kinetic analyses with possible physiological substrates indicated that all of the AtHOLs have low activities toward chloride. AtHOL1 was highly reactive to thiocyanate (NCS(-)), a pseudohalide, synthesizing methylthiocyanate (CH(3)SCN) with a very high k(cat)/K(m) value. We demonstrated in vivo that substantial amounts of NCS(-) were synthesized upon tissue damage in Arabidopsis and that NCS(-) was largely derived from myrosinase-mediated hydrolysis of glucosinolates. Analyses with the T-DNA insertion Arabidopsis mutants (hol1, hol2, and hol3) revealed that only hol1 showed increased sensitivity to NCS(-) in medium and a concomitant lack of CH(3)SCN synthesis upon tissue damage. Bacterial growth assays indicated that the conversion of NCS(-) into CH(3)SCN dramatically increased antibacterial activities against Arabidopsis pathogens that normally invade the wound site. Furthermore, hol1 seedlings showed an increased susceptibility toward an Arabidopsis pathogen, Pseudomonas syringae pv. maculicola. Here we propose that AtHOL1 is involved in glucosinolate metabolism and defense against phytopathogens. Moreover, CH(3)Cl synthesized by AtHOL1 could be considered a byproduct of NCS(-) metabolism.