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

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.


Supplementary Information S1. Methodology of chemical synthesis.
A. 2AP 2-(Methoxycarbonyl)-1-pyrroline (6) Following a method identical to that of De Kimpe (1993), To a solution of methyl prolinate 7 (1.94 g, 15.1 mmol) in anhydrous ether (130 mL) at 0 ºC, was added drop wise tert-butylhypochlorite (1.67 g, 15.4 mmol) and the reaction then stirred for 1 hour. The reaction was checked by GC-MS to monitor for the formation of the N-chloro product, then triethylamine (1.55 g, 15.4 mmol) was added drop wise and stirred at room temperature overnight. The mixture was filtered through a bed of celite then concentrated in vacuo to afford an oil which was purified by short-path Kugelrohr distillation 13 mmHg]) to give 2-(methoxycarbonyl)-1-pyrroline (244.7 mg, 13%) as a colourless oil with spectroscopic data identical to those reported in the literature by De Kimpe (1993).
2-Acetyl-1-pyrroline (1) and 6-methyl-5-oxo-2,3,4,5-tetrahydropyridine (5) Synthesised using a method similar to that of De Kimpe (1993): To a solution of 6 (45 mg, 3.5 mmol) in anhydrous ether (5 mL) under nitrogen at -15 ºC (salt / ice) was added methylmagnesium bromide (140 µL, 4.0 mmol, 3 M solution in ether) dropwise over 5 mins. The reaction was monitored over 2.5 hours by GC-MS, then quenched with 1 M HCl and stirred for 30 min. The ethereal layer was then removed and discarded and the aqueous layer neutralised with 2 M NaOH and basified to pH 10. The mixture was extracted with DCM (5 × 5 mL), dried over anhydrous sodium sulfate, filtered and stored in the freezer in DCM. The crude residue was examined by GC-MS and by 1 H NMR, revealing the presence of traces of the double addition product. A portion of the final product mixture was purified by silica flash chromatography using silica that was pre-washed with hexane/triethylamine (10 mL, 99:1). After application of the product mixture to the column, fractions were eluted with ether:pentane (1:3), combined and concentrated to give a mixture of 2AP and 6M5OTP, together with trace amounts of tertiary alcohol by-product, as a malodorous slightly pale yellow oil. This was then carefully concentrated under nitrogen, diluted in CDCl 3 and and subjected to acid-base partitioning by extraction into 1 M HCl, followed by basification to pH 10 using 1 M NaOH, and extraction back into deuterated chloroform. GC-MS and 1 H NMR data were run, showing a ratio of 76:24 (GC-MS) and 78:22 ( 1 H NMR). The remaining unpurifed sample was stored frozen in solution in DCM at -78 ºC.

B. 1-Pyrroline (4)
Following a method identical to that of Ottinger and Hofmann (2002): To a solution of L-proline (2.58 g, 22.4 mmol) in water (40 mL), was added to an aqueous solution of sodium metaperiodate (50 mL, 0.3 mmol/L) and the reaction then stirred for 2 hours in the dark at room temperature. The pH of the reaction was then adjusted to 9 with 1 M aqueous sodium hydroxide. The solution was extracted with diethyl ether (3 × 20 mL), and the combined organic layers then washed with brine (20 mL).
After the organic layer was dried over Na 2 SO 4 it was concentrated under a gentle stream of nitrogen.
A portion of the product mixture was carefully concentrated under nitrogen and rapidly purified on a neutral alumina pipette column (slightly deactivated, pre-washed with n-pentane/water; 5 mL, 99:1).

Nuclear Magnetic Resonance Spectroscopy
Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded on a Bruker Avance 500 spectrometer using a 5 mm SEI probe or a Bruker Avance DRX 700 spectrometer with a 5 mm TXI Zgrad probe. Carbon-13 nuclear magnetic resonance ( 13 C NMR) spectra were recorded on a Bruker Avance DRX 700 spectrometer with a 5 mm TXI Zgrad probe. Measurements were made in deuterated chloroform (CDCl 3 , referenced to: δ H 7.26 ppm, δ C 77.16 ppm). Chemical shifts (δ) were recorded in parts per million (ppm) and coupling constants (J values) were measured in Hertz (Hz).
Two dimensional NMR (2D NMR) data were acquired from Bruker Avance 500 and 700 MHz instruments. Gradient enhanced HMBC (geHMBC) and HSQC (geHSQC) NMR were obtained with 8 to 64 transients per increment with the evolution delay set at n J CH of 4 Hz or 8 Hz (geHMBC) and 1 J CH of 135 Hz (geHSQC). Gradient COSY (gCOSY) was recorded with 8 to 32 transients per increment with a pulse delay of 2.0 seconds.

Gas Chromatography/Mass Spectrometry
Gas chromatography/mass spectrometry (GC/MS) spectra were recorded on a Shimadzu GC-MS-QP2010 Plus. GC/MS programme: flow rate 1.5 mL/min; initial oven temperature 100 o C (isothermal for 3 minutes); ramped 16 o C/min to 270 o C held for 10 min; injection temperature 250 o C.

Comprehensive Two-dimensional Gas Chromatography/Time-of-flight Mass Spectrometry
Synthesised authentic standards (1 µL) were allowed to equilibrate in 10ml GC vials for two hours without heating and agitation. The headspace (500 µL) was collected using a 2.5 mL headspace syringe and injected on a Leco Pegasus 4D GC × GC-TOF-MS (St. Joseph, MI, USA) in splitless mode. The inlet and transfer line temperatures were set at 250 and 240 o C, respectively. The primary oven temperature was held initially at 45 o C for 1 min and then ramped at a rate of 10 o C/min to 235 o C. The secondary oven and the modulator were set at 15 and 25 o C higher than the primary oven, respectively for the entire run. The modulation period was set at 2.5 s, with a hot pulse of 0.4 s and 0.85 s cold pulse between stages. Data acquisition was done in full scan at 1500 V and 70 V electron energy. The ion source was set at 240 o C. TOF-MS autotuning (acquisition system adjustment, ion optic focusing and mass calibration) was done every 48 hours. The total analysis time is 30 mins.