Comparative omics reveals unanticipated metabolic rearrangements in a high-oil mutant of plastid acetyl-CoA carboxylase

Heteromeric acetyl-CoA carboxylase (ACCase) catalyzes the ATP-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, the committed step for de novo fatty acid synthesis. In plants, ACCase activity is controlled at multiple levels, including negative regulation by biotin attachment domain-containing (BADC) proteins, of which the badc1/3 double mutant leads to increased seed triacylglycerol accumulation. Unexpectedly, the Arabidopsis badc1/3 mutant also accumulates more protein. The metabolic consequences from both higher oil and protein were investigated in developing badc1/3 seed using global transcriptomics, translatomics, proteomics, metabolomics and other biomass measurements. Changes included increased storage proteins and lipid-droplet packaging proteins, increased SDP1 lipase, altered organic acid metabolism, and reduced extracellular lipid synthesis perhaps offsetting the increase in TAG. We present a model of how Arabidopsis adapted to deregulated ACCase, resulting in more oil, and altered flux through pathways that partition carbon and propose targets for future bioengineering of seed storage reserves. To investigate the matabolic consequences of increased oil content due to knocking out genes BADC1 and BADC3 in Arabidopsis. We perfomed gene expression analysis using data from Arabidopsis deveoping seeds colected at 9-10 days after flowering from a badc1/badc3 double knockout line compared to wild-type (Col0) line.Total RNA was purified using the NucleoSpin® RNA Plant kit (MACHEREY-NAGEL), as recommended by the manufacturer. Raw Illumina sequencing data was processed using the Cufflinks suite. Reads were mapped to the Arabidopsis reference genome, and gene expression was analyzed. We also performed translatomics analysis using plants expressing a transgene, HF-RPL18, tagged with a 6X histidine-FLAG epitope, driven by the glycinin promoter from soybean. The HF-RPL18 gene was cloned into the pBinGlyRed3 vector and transformed into Agrobacterium tumefaciens, which was then used to transform wild-type (Col-0) and badc1/3 plants via the floral dip method. T1 seeds were screened for DsRed fluorescence and RPL18 protein expression using an Anti-FLAG antibody. For ribosome-associated RNA enrichment, samples were incubated with EZ view Red Anti-FLAG M2 Affinity Gel to isolate the FLAG-tagged ribosomes. RNA was extracted from the beads using a miniprep kit. For RNA-Seq and TRAP-Seq data analysis, raw Illumina sequencing data was processed using the Cufflinks suite. Reads were mapped to the reference genome, and gene expression was analyzed. Translation efficiency (TE) was calculated by comparing TRAP-Seq reads to total RNA-Seq reads, with significant changes identified through Z-score normalization.