The branched-chain amino acid-related isoleucic acid: recent research advances

Abstract

Isoleucic acid (ILA) was identified in human patients with maple syrup urine disease (MSUD) half a century ago. MSUD patients, who are defective in the catabolism of branched-chain amino acids (BCAAs), that is, isoleucine, leucine, and valine, have urine with a unique maple syrup odour related to the accumulation of BCAA breakdown products, largely 2-keto acid derivatives and their reduced 2-hydroxy acids including ILA. A decade ago, ILA was identified in Arabidopsis thaliana. Subsequent studies in other plant species indicated that ILA is a ubiquitously present compound. Since its identification in plants, several efforts have been made to understand the biological significance and metabolic pathway of ILA. ILA plays a positive role in plant signalling for defence responses against bacterial pathogens by increasing the abundance of salicylic acid aglycone through competitive inhibition of SA deactivation by glucosylation. Here, we review recent progress in the characterization of ILA biosynthesis and function in plants and discuss current knowledge gaps and future directions in ILA research.

Keywords: 2‐hydroxy acid; 2‐keto acid; BCAT; BCKDH; ILA; KMVA.

Fig. 1

Chemical structure of the branched‐chain amino acid isoleucine and the related 2‐oxo‐methylvaleric acid (2‐KMVA), being the first product of Ile catabolism and the immediate biosynthetic precursor of Ile. 2‐hydroxy‐3‐methylvaleric acid (ILA) is the reduced form of 2‐KMVA.

Fig. 2

Putative subcellular localization of ILA biosynthesis in plants. In the chloroplast, 2‐KMVA is an immediate precursor for Ile biosynthesis, and in the presence of a reductase, 2‐KMVA could be reduced to ILA (dashed green lines). In the cytosol and in mitochondria, 2‐KMVA is the first product in the catabolism of Ile by transaminases. In the presence of appropriate reductases, 2‐KMVA could be reduced to ILA (dashed green arrows). The dashed green lines are all hypothetical enzymatic steps.

Fig. 3

(a) ILA content in 3‐week‐old shoots of various Arabidopsis mutant lines compared to the wild type. Leaf samples were collected from soil‐grown plants for ILA quantification. ILA content was measured in leaf extracts after anion‐exchange solid‐phase extraction (SPE) followed by silylation and GC‐MS analysis (Maksym et al. 2018). Values represent the average of three biological replicates; error bars indicate the standard error of means. Statistical difference compared to wild type (Benjamini‐Hochberg adj. P‐values **, <0.01; *, <0.05; n.s., non‐significant; ANOVA, Holm‐Sidak post‐hoc method). The experiment was repeated independently and with comparable statistical results. Abbreviations: WT, wild type; l‐ldh, l ‐lactate dehydrogenase mutant (Alonso et al. 2003); d‐ldh, d ‐lactate dehydrogenase mutant; gox1, glyoxylate oxidase 1; haox1, hydroxy acid oxidase 1; haox2, hydroxy acid oxidase 2; omr1, L‐O‐methythreonine resistance 1, threonine deaminase gain of function mutant (Mourad & King 1995). (b) ILA content in shoots of 4‐week‐old soil‐grown Arabidopsis plants irrigated once with 500 μM ILA. ILA was analysed 3 days after its application. Values are the average of four biological replicates. Error bars are standard error of means (**P < 0.01, ANOVA).

Fig. 4

Qualitative assessment of putative ILA glucoside (m/z 293.1242, C₁₂H₂₁O₈, negative mode) by ultrahigh‐resolution Fourier transform ion cyclotron resonance mass spectrometry. Arabidopsis lyrata, Brassica oleracea subsp. oleracea, Brassica napus, Solanum lycopersicum cv. ‘Micro‐Tom’, Nicotiana tabacum, Hordeum vulgare L. cv. Barke, Zea mays, and Populus spp. were grown in a greenhouse with 10 h light at 24°C and at 18°C during the dark period with 60% relative humidity. Leaf samples (n = 5; mean ± SD) were collected at young seedling stage, extracted and analysed as described previously (von Saint Paul et al. 2011).

Fig. 5

Summary of the differences in ILA biosynthesis, regulation, and functional roles in plants and mammals.