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.