Dynamic regulatory control of plant heteromeric ACCase revealed by spatiotemporal stoichiometry

Despite being comprised of four catalytic subunits and affected by three classes of regulatory proteins, the plastid heteromeric acetyl-CoA carboxylase (hetACCase) remains poorly understood from both structural and protein stoichiometry perspectives. Here, we expanded an AQUA-multiple reaction monitoring (AQUA-MRM) assay to quantify the absolute abundance of hetACCase catalytic subunits and regulatory effectors across Arabidopsis organs, developmental stages, and light conditions. Our results demonstrate that the in vivo stoichiometry of ACCase catalytic subunits and regulatory effectors varies with organ, light, and development. The catalytic subunits to ACCase were detected in all organs under all conditions, unlike most heterotrophs which often only conditionally express ACCase. The β-CT subunit accumulated at higher quantities than its three catalytic counterparts, possibly because it is plastid encoded. Highest average abundance of β-CT was observed in 11 DAF developing seed (112 fmol/µg), followed by young leaves (54.7 fmol/µg), 7 DAF pod walls (17.1 fmol/µg), and roots (16.8 fmol/µg). Notably, catalytic subunits BC, BCCP (summed isoforms), and α-CT exhibited nearly 1:1:1 in vivo stoichiometry throughout the plant. Maximum average abundances of BC, BCCP, and α-CT were 95.1, 63.4, and 79.6 fmol/µg in developing seeds, 34.0, 26.5, and 29.1 fmol/µg in leaves, 12.1, 8.6, and 8.4 fmol/µg in pod walls, and 8.5, 6.0, and 6.4 fmol/µg in roots, respectively, indicating structural homogeneity and coordinated expression. In contrast, regulatory effectors, BADC, CTI, and PII did not obey any consistent stoichiometric relationship with catalytic subunits, often exceeding them in abundance, but in unique and specific ways suggesting adaptive roles for each effector class to sense and regulate a ubiquitously-expressed plant ACCase. Highest levels of both PII and BADC were observed in developing seed and pod walls, with PII peaking at 15 DAF seed (550 fmol/µg) and BADC (summed isoforms) reaching 239 fmol/µg at 11 DAF. In pod walls, BADC (42.5 fmol/µg) exceeded PII (27.6 fmol/µg) at 7 DAF. In leaves, BADC (119 fmol/µg in young leaves) and CTI (98.5 fmol/µg in mature leaves) were predominant, whereas roots were characterized primarily by BADC accumulation (34.8 fmol/µg). Increases observed after light onset in hetACCase catalytic subunits and regulatory effectors were most pronounced in leaves, followed by pod walls, and developing seeds, whereas roots displayed weak or inverse responses. Together, these findings redefine plastid hetACCase as a dynamic metabolic hub that integrates developmental, metabolic, and environmental signals through tissue-specific regulatory modules, including PII, CTIs, and BADCs, to coordinate fatty acid biosynthetic flux; a strategy that attenuates activity in response to cellular cues.