Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis

Abstract

Citrate, a major determinant of citrus fruit quality, accumulates early in fruit development and declines towards maturation. The isomerization of citrate to isocitrate, catalyzed by aconitase is a key step in acid metabolism. Inhibition of mitochondrial aconitase activity early in fruit development contributes to acid accumulation, whereas increased cytosolic activity of aconitase causes citrate decline. It was previously hypothesized that the block in mitochondrial aconitase activity, inducing acid accumulation, is caused by citramalate. Here, we investigated the effect of citramalate and of another aconitase inhibitor, oxalomalate, on aconitase activity and regulation in callus originated from juice sacs. These compounds significantly increased citrate content and reduced the enzyme's activity, while slightly inducing its protein level. Citramalate inhibited the mitochondrial, but not cytosolic form of the enzyme. Its external application to mandarin fruits resulted in inhibition of aconitase activity, with a transient increase in fruit acidity detected a few weeks later. The endogenous level of citramalate was analyzed in five citrus varieties: its pattern of accumulation challenged the notion of its action as an endogenous inhibitor of mitochondrial aconitase. Metabolite profiling of oxalomalate-treated cells showed significant increases in a few amino acids and organic acids. The activities of alanine transaminase, aspartate transaminase and aspartate kinase, as well as these of two γ-aminobutyrate (GABA)-shunt enzymes, succinic semialdehyde reductase (SSAR) and succinic semialdehyde dehydrogenase (SSAD) were significantly induced in oxalomalate-treated cells. It is suggested that the increase in citrate, caused by aconitase inhibition, induces amino acid synthesis and the GABA shunt, in accordance with the suggested fate of citrate during the acid decline stage in citrus fruit.

Mutations in the tricarboxylic acid cycle enzyme, aconitase 2, cause either isolated or syndromic optic neuropathy with encephalopathy and cerebellar atrophy

“…Aconitase activity of whole cell extracts and mitochondria-enriched fractions from control fibroblasts was progressively inhibited using increasing concentrations of citramalate (maximum inhibition at 80–100 mM, see online supplementary figure S2A). ACO1 activity remained unchanged even at high concentration of citramalate, demonstrating that citramalate selectively inhibits ACO2 in human cultured skin fibroblasts, as shown in plants 6. The use of citramalate allowed to directly measure the ACO2 and ACO1 activities in whole cell extracts from control and patients’ fibroblasts.…”

“…Aconitase activity (ie, consumption of cis -aconitate) was coupled to that of native isocitrate dehydrogenase, at 340 nm, in the presence of 0.8 mM Nicotinamide adenine dinucleotide (NAD), 0.8 mM Nicotinamide adenine dinucleotide phosphate (NADP) and 0.10 mM MgCl 2 . The reaction was initiated by adding 5 mM cis -aconitate to the medium, either in absence or in the presence of 100 mM potassium citramalate monohydrate (Sigma Aldrich), a potent inhibitor of ACO2 in citrus fruit 6. Protein amounts were quantified using Bradford reagent (BioRad).…”

Evolution and Functional Implications of the Tricarboxylic Acid Cycle as Revealed by Phylogenetic Analysis

“…This fact coupled with the central point of this enzyme in the regulation of organic acid metabolism, aconitase seems to be a potential and suitable target for metabolic engineering applications. For instance, the recent combination of genetic and molecular approaches has demonstrated the crucial role of this enzyme in controlling organic acid content in ripe tomato fruit (Morgan et al 2013), whereas in citrus fruit it was possible to manipulate the fruit acidity by changes in amino acid metabolism (Degu et al 2011). …”

Integration of Metabolomics and Subcellular Organelle Expression Microarray to Increase Understanding the Organic Acid Changes in Post‐harvest Citrus Fruit

“…At the later stage of fruit development and maturity, the γ‐aminobutyric acid (GABA) shunt could effectively reduce cytosolic acidity by catabolizing the glutamate produced as a product of citrate metabolism (Cercós et al ). When the aconitase inhibitor (citramalic acid) was used to treat citrus callus, it was found that excessive citric acid was transformed into amino acids via the GABA shunt (Degu et al ). The above‐mentioned studies showed that the GABA shunt is involved in the regulation of organic acid metabolism.…”