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Heteromeric acetyl‐CoA carboxylase (htACCase) catalyzes the committed step ofde novofatty acid biosynthesis in most plant plastids. Plant htACCase is comprised of four subunits: α‐ and β‐carboxyltransferase (α‐ and β‐CT), biotin carboxylase, and biotin carboxyl carrier protein. Based onin vivoabsolute quantification of htACCase subunits, α‐CT is 3‐ to 10‐fold less abundant than its partner subunit β‐CT in developing Arabidopsis seeds [Wilson and Thelen,J. Proteome Res., 2018, 17 (5)]. To test the hypothesis that low expression of α‐CT limits htACCase activity and flux through fatty acid synthesisin planta, we overexpressedPisum sativum α‐CT, either with or without its C‐terminal non‐catalytic domain, in bothArabidopsis thalianaandCamelina sativa.First‐generation Arabidopsis seed of35S::Ps α‐CT(n = 25) and35S::Ps α‐CT^Δ406‐875(n = 47) were on average 14% higher in oil content (% dry weight) than wild type co‐cultivated in a growth chamber. First‐generation camelina seed showed an average 8% increase compared to co‐cultivated wild type. Biochemical analyses confirmed the accumulation ofPsα‐CT andPsα‐CT^Δ406‐875protein and higher htACCase activity in overexpression lines during early seed development. OverexpressedPsα‐CT co‐migrated with nativeAtβ‐CT during anion exchange chromatography, indicating co‐association. By successfully increasing seed oil content upon heterologous overexpression of α‐CT, we demonstrate how absolute quantitation ofin vivoprotein complex stoichiometry can be used to guide rational metabolic engineering.

12-hydroxy-jasmonoyl-isoleucine (12OH-JA-Ile) is a metabolite in the catabolic pathway of the plant hormone jasmonate, and is synthesized by the cytochrome P450 subclade 94 enzymes. Contrary to the well-established function of jasmonoyl-isoleucine (JA-Ile) as the endogenous bioactive form of jasmonate, the function of 12OH-JA-Ile is unclear. Here, the potential role of 12OH-JA-Ile in jasmonate signaling and wound response was investigated. Exogenous application of 12OH-JA-Ile mimicked several JA-Ile effects including marker gene expression, anthocyanin accumulation and trichome induction in Arabidopsis thaliana. Genome-wide transcriptomics and untargeted metabolite analyses showed large overlaps between those affected by 12OH-JA-Ile and JA-Ile. 12OH-JA-Ile signaling was blocked by mutation in CORONATINE INSENSITIVE 1. Increased anthocyanin accumulation by 12OH-JA-Ile was additionally observed in tomato and sorghum, and was disrupted by the COI1 defect in tomato jai1 mutant. In silico ligand docking predicted that 12OH-JA-Ile can maintain many of the key interactions with COI1-JAZ1 residues identified earlier by crystal structure studies using JA-Ile as ligand. Genetic alternation of jasmonate metabolic pathways in Arabidopsis to deplete both JA-Ile and 12OH-JA-Ile displayed enhanced jasmonate deficient wound phenotypes and was more susceptible to insect herbivory than that depleted in only JA-Ile. Conversely, mutants overaccumulating 12OH-JA-Ile showed intensified wound responses compared with wild type with similar JA-Ile content. These data are indicative of 12OH-JA-Ile functioning as an active jasmonate signal and contributing to wound and defense response in higher plants.