AtGH3.10 and JAR1 Produce 12-Hydroxyjasmonoyl-l-isoleucine from 12-Hydroxyjasmonic Acid in Arabidopsis thaliana

Jasmonates are plant hormones that regulate plant defense and development. 7-iso-Jasmonoyl-l-isoleucine (JA-Ile) is a representative active jasmonate which is biosynthesized from 7-iso-jasmonic acid (JA) by the jasmonoyl-amido synthases JASMONATE RESISTANT 1 (JAR1) and AtGH3.10 in Arabidopsis thaliana. 12-Hydroxy-7-iso-jasmonoyl-l-isoleucine (12-OH-JA-Ile) is another active jasmonate, and 12-hydroxylation of JA-Ile is considered the major biosynthetic pathway toward 12-OH-JA-Ile. Previous report elucidated that recombinant JAR1 showed a weak activity against 12-hydroxy-7-iso-jasmonic acid (12-OH-JA). However, the direct conversion from 12-OH-JA to 12-OH-JA-Ile in planta and the enzyme activity of AtG3.10 against 12-OH-JA have never been reported. Herein, a feeding experiment with deuterated 12-OH-JA confirms the direct conversion of 12-OH-JA to 12-OH-JA-Ile in wild-type Arabidopsis plants. The conversion from 12-OH-JA to 12-OH-JA-Ile is not observed in jar1 gh3.10 double mutant, suggesting that 12-OH-JA is converted to 12-OH-JA-Ile by JAR1 and AtGH3.10. Notably, enzyme assays show that the catalytic efficiency with 12-OH-JA for AtGH3.10 is higher than those with JA for AtGH3.10 and with 12-OH-JA for JAR1. Comparative analysis of JAR1 and AtGH3.10 structures and site-directed mutation analysis reveals that Ser120 in AtGH3.10 is the key amino acid residue responsible for its high catalytic efficiency against 12-OH-JA.