Metabolomics and genomics combine to unravel the pathway for the presence of fragrance in rice

Abstract

Since it was first characterised in 1983, 2-acetyl-1-pyrroline (2AP) has been considered to be the most important aroma compound in rice. In this study, we show four other amine heterocycles: 6-methyl, 5-oxo-2,3,4,5-tetrahydropyridine (6M5OTP), 2-acetylpyrrole, pyrrole and 1-pyrroline, that correlate strongly with the production of 2AP, and are present in consistent proportions in a set of elite aromatic rice varieties from South East Asia and Australia as well as in a collection of recombinant inbred lines (RILs) derived from indica Jasmine-type varieties, Australian long grain varieties (temperate japonica) and Basmati-type rice (Grp V). These compounds were detected through untargeted metabolite profiling by two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOF-MS), and their identity were confirmed by comparison with authentic standards analysed using gas chromatography mass spectrometry (GC-MS) and High Resolution GC × GC-TOF-MS (GC × GC HRT-4D). Genome-wide association analysis indicates that all compounds co-localised with a single quantitative trait locus (QTL) that harbours the FGR gene responsible for the production of GABA. Together, these data provide new insights into the production of 2AP, and evidence for understanding the pathway leading to the accumulation of aroma in fragrant rice.

Figure 1

PCA biplot of the volatile metabolites detected in rice by GC × GC-TOF-MS. Metabolites are the triangles, overlayed with the rice varieties (circles). (A) Set 1 (n = 11), PC1 = 24.8% and PC2 = 17.8%. (B) Set 2 (n = 341) PC2 = 7.91% and PC3 = 6.1%.

Figure 2

Identification of Analyte 5 as (6M5OTP). (A) GC × GC-TOF-MS 3D chromatogram of synthesised 2AP standard showing 2AP (RT: 627.5 s, 1.025 s) and 6M5OTP (RT: 707.5 s, 1.27 s). MS Electron ionisation fragmentation pattern of Analyte 5 detected by GC × GC-TOF-MS (B) synthesised 2 AP standard, and (C) rice. (D) GC-MS trace comparison of ratio between 2AP and 6M5OTP before (above) and after acid-base extraction (below). (E) Expansion of the¹H NMR of 2AP (red) and 6M5OTP (black). Solvent peaks are denoted in X.

Figure 3

GWAS of 2AP and linked compounds. (A) Physical distribution of the most significant SNPs in chromosome 8. The functional markers for FGR (diamonds) outperformed the MS SNPs (S8_19555833) for each of the five compounds. Lines connecting (A,B) denote the position of the S8_19555833 (previously MS SNP, red line) and the SNPs closest to FGR (SNPs S8_20311425 and S8_20408314, blue lines) relative to FGR (orange arrow). (B) Representative haplotypes of the population. Red cells represent SNPs identical to the aromatic line KDML 105, heterozygotes are represented by half-filled cells, while grey are missing data. The number of lines carrying each haplotype is indicated in the table. Asterisk denotes the haplotypes where linkage breaks between the MS SNP S8_19555833 are not predictive of the presence or absence of aroma, explaining the lower LOD score of SNP S8_19555833. (C) Zoom in of the QTL region on chromosome 1. The most significant SNPs (MS SNPs) are denoted in red. Location of the candidate genes are shown by dotted lines. (D) Box plot for the 2AP levels at different alleles of the MS SNP S1_42480486. The middle line indicates the median, the box indicates the range of the 25th to 75th percentiles of the total data, the whiskers indicate the interquartile range and the outer dots are outliers. n shows the number of samples used to make the plot over the total number of samples, p and α denote the p-values and alpha, respectively.

Figure 4

Chemical formation of 6M5OTP through in vitro acid/base workup. 2AP ring opens in excess acid to form an unstable diketone, which cyclises back to 2AP or to 6M5OTP under basic conditions.

Figure 5

Putative biosynthesis of 2AP. Enzymes are in italics: CuAO – copper amine oxidase; PAO – polyamine oxidase; AADH – amino aldehyde dehydrogenase; PDC – Pyruvate decarboxylase; PDH – pyruvate dehydrogenase complex. (A) TPP-catalysed transfer of a C2 unit to 4-aminobutanal. The reaction with 4-aminobutanal forms an unstable diketone 6-amino2,3-hexadione, which cyclises to either 2AP or 6M5OTP. (B) Cellular localisation of the reactions leading to the formation of 2AP and 6M5OTP.