ATP citrate lyase activity is post‐translationally regulated by sink strength and impacts the wax, cutin and rubber biosynthetic pathways

Abstract

SUMMARYCytosolic acetyl-CoA is involved in the synthesis of a variety of compounds, including waxes, sterols and rubber, and is generated by the ATP citrate lyase (ACL). Plants over-expressing ACL were generated in an effort to understand the contribution of ACL activity to the carbon flux of acetyl-CoA to metabolic pathways occurring in the cytosol. Transgenic Arabidopsis plants synthesizing the polyester polyhydroxybutyrate (PHB) from cytosolic acetyl-CoA have reduced growth and wax content, consistent with a reduction in the availability of cytosolic acetyl-CoA to endogenous pathways. Increasing the ACL activity via the over-expression of the ACLA and ACLB subunits reversed the phenotypes associated with PHB synthesis while maintaining polymer synthesis. PHB production by itself was associated with an increase in ACL activity that occurred in the absence of changes in steady-state mRNA or protein level, indicating a post-translational regulation of ACL activity in response to sink strength. Over-expression of ACL in Arabidopsis was associated with a 30% increase in wax on stems, while over-expression of a chimeric homomeric ACL in the laticifer of roots of dandelion led to a four-and two-fold increase in rubber and triterpene content, respectively. Synthesis of PHB and over-expression of ACL also changed the amount of the cutin monomer octadecadien-1,18-dioic acid, revealing an unsuspected link between cytosolic acetyl-CoA and cutin biosynthesis. Together, these results reveal the complexity of ACL regulation and its central role in influencing the carbon flux to metabolic pathways using cytosolic acetyl-CoA, including wax and polyisoprenoids.

Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria

“…Our results are not sufficiently comprehensive to enable us to provide a precise mechanism by which the observed changes generated in the trxo1 mutant could modulate the activity of a cytosolic enzyme. However, they are in keeping with data from a recent study suggesting posttranslational regulation of ACL (58).…”

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

“…The full‐length Tk1‐FEH cDNA was amplified using oligonucleotides 1‐FEH‐XhoI‐fwd and 1‐FEH‐XbaI‐rev (Table S3) and inserted into the expression vector pLab12.10 using the restriction sites XhoI and XbaI (Xing et al ., 2014). The final construct (pLab12.10‐CaMV35SP‐Tk1‐FEH‐CaMV35ST) was validated by sequencing.…”

“…Hexane phases were pooled and evaporated to dryness, and the residue was dissolved in 1 mL acetone overnight before analysis by GC‐MS as described by Xing et al . (2014). …”

Multi-Omics of Tomato Glandular Trichomes Reveals Distinct Features of Central Carbon Metabolism Supporting High Productivity of Specialized Metabolites

“…Acetyl-CoA, however, cannot be transported across the mitochondrial membrane. The supply of acetyl-CoA in the cytosol can be achieved via the citrate shuttle (Oliver et al, 2009;Xing et al, 2014). Citrate is transported from the mitochondria or the peroxisomes into the cytosol and cleavage of citrate by the cytosolic ATP-citrate lyase produces oxaloacetate and acetyl-CoA.…”

“…ATP-citrate lyase is strongly enriched in trichomes (Figure 9), as are a number of enzymes typically associated with this shuttle system. Recently, Xing et al (2014) highlighted the central role of ATP-citrate-lyase in the production of cutin and acetyl-CoA-derived polyhydroxybutyrate in transgenic Arabidopsis. Thus, the overexpression ATP-citrate lyase together with a number of enzymes involved in this process constitutes a potential engineering target for increasing flux through the MEV pathway.…”