Food Chemistry 436 (2024) 137788

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Integration of transcriptome, volatile and non-volatile metabolite profile

reveals characteristic aroma formation in Toona sinensis
Beibei Zhang a, b, d, Lifang Hao a, d, Jing Zhang c, Jinze Feng a, d, Cheng Wang a, d, *,
Jingfang Zhang a, d, *
a
College of Forestry, Northwest A&F University, Yangling, Shaanxi, 712100, China
b
Key Laboratory of Food Safety Risk Assessment, School of Chemistry Engineering, Xian University, Xian, Shaanxi, 710065, China
c
College of Horticulture, Northwest A&F University, Yangling, Shaanxi, 712100, China
d
Shaanxi Key Laboratory of Economic Plant Resources Development and Utilization, Yangling, Shaanxi, 712100, China

A R T I C L E I N F O A B S T R A C T

Keywords: Toona sinensis is renowned for its unique aroma, but the formation mechanism remains unclear. In this study,
Toona sinensis volatile and non-volatile metabolites were combined with transcriptomes to investigate the potential mechanism
Volatile metabolite of aroma formation in T. sinensis buds (TSB) and microgreens (TSM). Volatile sulfur compounds (VSCs) and
Non-volatile metabolite
terpenes were the main volatiles of TSM and TSB, respectively. 20 volatiles were identified as potential bio­
Aroma profile
Transcriptome
markers, mainly VSCs and terpenes. In VSC biosynthesis pathways, cysteine was primarily synthesized from
serine transformation in TSM. S-(trans-L-propenyl)-L-cysteine was likely to be the main precursor of VSC
biosynthesis in T. sinensis. Higher expression of lachrymatory-factor synthase (LFS) consuming more precursor
(1-propenyl sulfenic acid) in TSB led to reduced accumulation of VSCs. Isopentenyl diphosphate isomerase (IDI)
and mevalonate diphosphate decarboxylase (MPDC) might play crucial roles in T. sinensis terpene biosynthesis.
This study provided valuable insights into the formation of characteristic aromas in T. sinensis.

1. Introduction
Toona sinensis (A. Juss.) Roem (Meliaceae) is a widely cultivated
plant in eastern and southeastern Asia (Peng et al., 2019). In China, it is
one of the most important traditional medicine plants with a history of
over 2000 years and its multiple physiological effects have been proven
in modern research, such as anti-inflammatory, anti-diabetic and anti­
bacterial activities (Chen et al., 2017; Chen et al., 2022 Dong et al.,
2013; Edmonds and Staniforth, 1998; Wu et al., 2010). Additionally,
T. sinensis buds (TSB) are highly sought-after woody vegetables
renowned for the unique aroma (Chen et al., 2022; Shen et al., 2019;
Wang et al., 2023a; Yang et al. 2011). However, TSB can only be har­
vested during a brief three-week period in spring and quickly becomes
lignified, leading to its high prices and inability to meet dietary demands
(Wang et al., 2023a; Zhao et al., 2021). Despite attempts to alleviate this
problem by developing processed TSB products such as frozen, dried,
blanched, and salted, their aroma clearly differed from that of fresh TSB
(Wang et al., 2020; Zhai & Granvogl, 2019; Zhang et al., 2022).
Currently, T. sinensis microgreens (TSM) are cultivated by hydroponics
using T. sinensis seeds, possessing a fresher aroma that is more similar to
TSB (Ding et al., 2023; Sharma et al., 2022). TSM can be cultivated
throughout the year with a short production cycle of approximately
three weeks (Ding et al., 2023; Li et al., 2013). Therefore, TSM was
considered as an ideal aroma substitute for fresh TSB, particularly dur­
ing non-harvest periods. However, the characteristics of TSM aroma
have not been systematically studied until now.
As the key quality characteristics of T. sinensis, unique aroma of TSB
has been investigated by several research groups. Currently, most
studies focus on the differences in volatile organic compounds (VOCs)
between different varieties (or origins) and various processing treat­
ments (Liu et al., 2013; Yang et al., 2019; Zhai & Granvogl, 2020a;
2020b). Over 200 VOCs have been identified in raw and processed TSB
samples, including volatile sulfur compounds (VSCs), terpenoids, alde­
hydes, alcohols, ketones, and esters (Liu et al., 2013; Yang et al., 2019;
Zhai & Granvogl, 2019). VSCs, such as 2-mercapto-3,4-dimethyl-2,3-
dihydrothiophene, 3,4-dimethyl-thiophene and 2,4-dimethyl-thiophene
were repeatedly identified as the main contributors to the characteristic
aroma of T. sinensis due to the lower odor thresholds and more

* Corresponding authors.
E-mail addresses: jmcookie@nwsuaf.edu.cn (C. Wang), zhangjf@nwsuaf.edu.cn (J. Zhang).

https://doi.org/10.1016/j.foodchem.2023.137788
Received 26 July 2023; Received in revised form 30 September 2023; Accepted 15 October 2023
Available online 17 October 2023
0308-8146/© 2023 Elsevier Ltd. All rights reserved.
B. Zhang et al. Food Chemistry 436 (2024) 137788

Fig. 1. The phenotypes of TSB (A) and TSM (B), and their radar chart (C) and the principal component analysis by E-nose (D).

2
B. Zhang et al.
distinctive odors (Wang et al., 2022; Zhai & Granvogl, 2019). Addi­
tionally, terpenoids, which showed high levels in T. sinensis, played
crucial roles to reconcile the irritating odor of sulfurous VSCs with their
sweet and floral aroma (Liu et al., 2013; Wang et al., 2023b; Zhang et al.,
2022). Hexanal, (E)-2-hexenal and (Z)-3-hexen-1-ol were recognized as
green notes offering grassy and leafy aroma in T. sinensis, which might
play the similar roles with terpenoids in the aroma formation of TSB
(Wang et al., 2023b).
Although the potential functions of different VOCs had been inves­
tigated in the characteristic aroma formation of T. sinensis, it was evident
that the profiles of these detected VOCs also varied significantly based
on the literature. For instance, TSB harvested from the same tree
exhibited differential compositions and contents of VOCs in different
years (Wang et al., 2023a). As a typical secondary metabolite, the aroma
formation of T. sinensis was affected by various factors, such as culti­
vated varieties, cultivated conditions, picking time, pests and disease.
Due to these factors, the potential mechanism of aroma formation in
T. sinensis (especially for VSCs) has not been clearly illuminated until
now. Compared with TSB, TSM can be cultivated in green house with
standardized production procedures. The stable and dependable condi­
tions of TSM production would be benefit for the thorough investigation
of the characteristic aroma formation of T. sinensis. However, there are
few reports on the aroma characteristics and intracellular formation of
TSM.
The main goals of this work are to clarify the aroma characteristics of
VOCs in TSM, investigate the differences in volatile and non-volatile
compounds during the aroma formation of TSB and TSM, as well as
explore the potential intracellular metabolic characteristics of the main
VOCs biosynthesis in TSB and TSM. The information would provide
valuable insights into the characteristic aroma formation and regulation
in T. sinensis.
2. Materials and methods
2.1. Chemicals and reagents
Chromatography-grade standards were purchased from Yuanye Bio
Co., Ltd. (Shanghai, China) including 2-nonanone and 2-chloro-L-
phenylalanine. N-alkane solution (C8 to C40) was purchased from Anpel
(Shanghai, China). The formic acid, methanol and acetonitrile were
purchased from Aladdin (Shanghai, China) and Merck (Darmstadt,
Germany). Methiin, γ-L-glutamyl-S-methyl-L-cysteine and S-(trans-1-
propenyl)-L-cysteine were synthesized by Siyuanyongtuo Technology
Co., Ltd. (Beijing, China) and the purity was at least 95 %. γ-L-glutamyl-
S-allyl-L-cysteine was purchased from USP (Rockville, MD, USA). S-allyl-
L-cysteine sulfoxide, S-allyl-L-cysteine, S-methyl-L-cysteine and 8 amino
acids were purchased from Alta Scientific Co., Ltd. (Tianjin, China).
Except for synthetic standards, all purchased standards were HPLC
grade with a purity of at least 98 %.
2.2. TSM generation
Based on our previous work on seven T. sinensis cultivars, the red
cultivar T. sinensis from Ankang, Shaanxi (108.90◦ E, 32.30◦ N) contains
the highest total and characteristic aroma, so the buds we picked and
seeds for the generation were both red cultivar T. sinensis from the same
geographical localization (Wang et al., 2023a).
The generation procedures were guided by a company that culti­
vated TSM for years, Xi’an Qingzhihe Agriculture and Forestry Tech­
nology Ltd. The dehydrated seeds with uniform size were sterilized by 1
% potassium permanganate at room temperature for 30 min and then
rinsed three times with sterilized water. Sterilized seeds were soaked
into deionized water at 26 ℃ with the mass ratio of 1:10 (seeds/water,
w/v) for 24 h in the dark, and then were transferred to a leakage hole
plastic box with a piece of wet seedling-growing paper at the bottom
(sterilized by 1 % potassium permanganate). The seeds were germinated
3
Food Chemistry 436 (2024) 137788
was at 26 ± 1 ◦ C for 14 days with a humidity of 70 % in dark. After that,
the microgreens were exposed to daylight at 26 ± 1 ◦ C for 7 days with a
humidity of 70 % in the plant factory in College of Forestry, Northwest
A&F University, Yangling, Shaanxi. Sterilized water was sprayed to keep
the seedling paper humid during generation.
2.3. Plant materials preparation
TSB were picked on April 15, 2022 (Fig. 1A). TSM were harvested in
10 cm in length in April of 2022 (Fig. 1B). After removing non-edible
parts, every 3 random buds (TSB) or boxes (TSM) were mixed and
ground in liquid nitrogen. 3 or 5 biological replicates of the samples
were taken for the analysis of E-nose and headspace solid-phase
microextraction-mass spectrometry coupled with gas chromatography-
mass spectrometry (HS-SPME-GC/MS) within a short time frame. The
ultra-performance liquid chromatography tandem mass spectrometry
(UPLC-MS/ MS), ultra-high-performance liquid chromatography tan­
dem mass spectrometry (UHPLC-MS/MS) and RNA extraction samples
were immediately frozen in liquid nitrogen and stored at − 80 ◦ C until
use.
2.4. E-nose analysis
The E-nose system (PEN3, Air sense Analytics GmbH, Germany) was
used to detect volatile compounds of TSB and TSM according to the
method described by our group before (Wang et al., 2023a). The system
consisted of 10 metal oxide sensors (W1C, W5S, W3C, W6S, W5C, W1S,
W1W, W2S, W2W and W3S) as shown in Table S1. Briefly, 0.2 g of TSB
and TSM samples were placed into a 20 mL vial, sealed with a PTFE-
coated cap, and then equilibrated at room temperature for 10 min.
Before the sample detection, the air washing time was set as 300 s. The
probe was inserted for measurement and lasted for 120 s. All samples
were run with five repetitions.
2.5. Extraction and determination of volatile metabolites
2.5.1. Extraction of volatile metabolites
The volatile metabolites of TSB and TSM were extracted by head­
space solid-phase microextraction (HS-SPME) described by Wang et al.
(2020). 2.0 g sample and 100 µL internal standard (2-nonanone of 1
ppm) were put into a 20 mL vial adding with 200 mL of saturated sodium
chloride solution to prevent enzymatic reactions. Immediately, vials
were capped with a tetrafluoroethylene rubber cushion cap and equili­
brated at 40 ◦ C for 10 min. Then, the extraction and adsorption were
performed by inserting the pretreated fiber (50/30 μm CAR/PDMS/
DVB, Supelco, Sigma, USA) into the headspace bottle for 15 min.
2.5.2. GC–MS analysis
The Thermo Trace Ultra GC (Waltham, MA, USA) system and a
Thermo ISQ MS (Waltham, MA, USA) were used for separation and
qualitative determination of the volatile metabolites. Two capillary
columns of different polarities, HP-Innowax (60 m/0.25 mm/0.25 µm)
and DB-5 (30 m/0.25 mm/0.25 µm), were fitted with the system
respectively. The carrier gas was 99.99 % helium gas with a flow rate of
1.0 mL/min. Choosing unsplit as the injection modes, the injector tem­
perature and ion source temperatures were set at 250 ◦ C. Mass spectra
were recorded at 70 eV, where the acquisition was full-scan mode with
the mass range of 40–500 amu. The oven temperature program was
optimized based on the method described by Wang et al. (2020) and the
details were listed in Table S2. Compounds were identified based on the
NIST-17 database library. A series of n-alkanes C8-C40 (Anpel laboratory
technologies, Shanghai) were used to determine RIs for each compound
on the two capillary columns.
2.5.3. Qualitative and quantitative analysis of volatile metabolites
The volatile compounds were qualitative by the following methods:
B. Zhang et al.
(A) raw ion fragmentation spectra acquired by GC–MS matched with MS
library NIST 17 and the SI or/and RSI score was over 800; (B) com­
pounds were recognized by both columns of different polarities and
could also meet the SI or/and RSI score was over 700; (C) the difference
between the calculated retention indices (RIc) and reference retention
indices (RIf) according to NIST database (https://webbook.nis t.gov) or
researches on volatiles of TSB by the non-isothermal Van Den Dool and
Kratz RI was less than 30 (van Den Dool & Dec. Kratz, 1963). Potent
compounds satisfying at least one of the three methods listed above were
considered as identified volatiles, which were otherwise removed.
The concentration of each volatile component was calculated by the
internal standard in HP-Innowax (Li et al., 2023). The calculation for­
mula was as follows:
Ci = (Mis × Ai )Ã⋅(Ais × M0 ) × fi × 1000.
Herein, Ci is the concentration of identified compound (µg/g), Mis
and M0 are the weight of internal standard (µg) and each detected TS
sample (g), Ai and Ais are the peak areas of the identified compound and
internal standard, fi is the correction factor of identified compounds,
which was regarded as 1.0 (Li et al., 2023).
2.6. Extraction and determination of non-volatile metabolites
2.6.1. Metabolite extraction
Metabolite extraction and metabolomic analysis were performed as
described by Zhang et al. (2022). 50 mg sample was added to 1 mL
extraction liquid (methanol: acetonitrile: water = 2:2:1 containing 20
mg/L internal standard (2-chloro-L-phenylalanine)). The samples were
homogenized in an automatic rapid grinder (JXFSTPRP-24, Jing Xin,
Shanghai, China) using a steel ball and ultrasonicated in ice water for 5
min. The samples were stored at − 20 ◦ C for 1 h and centrifugation at
12000 rpm for 15 min at 4 ◦ C. Then 500 µL of the supernatant was
transferred to a vial and concentrated to powder. With the addition of
160 µL of extract solution (volume ratio of acetonitrile to water = 1:1),
the suspension was vortexed for 30 s and redissolved by ultrasound for
10 min in an ice bath. Finally, the suspension was kept for 30 min at 4 ◦ C
before centrifugation (12000 rpm at 4 ◦ C for 15 min) to obtain super­
natants ready for injection. A quality control (QC) sample was prepared
by combining equal aliquots from each sample. The TSB and TSM
samples were extracted for metabolites in triplicate.
2.6.2. UPLC-MS/MS analysis
The UPLC-MS/MS system for non-volatile metabolome analysis is
composed of Waters ACQUITY UPLC I-Class PLUS system (Waters, USA)
tandem Waters Xevo G2-XS QTof high resolution mass spectrometer
(Waters, USA). The column used is purchased from Waters Acquity
UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm). The chromatographic
separation conditions were shown in Table S3 and the mass spectro­
metric conditions were as follows: The low collision energy is 2 V, the
high collision energy range is 10–40 V, and the scanning frequency is
0.2 s for a mass spectrum. Both positive and negative ion modes were
used for mass spectrometry detection. The parameters of the ESI ion
source are as follows: capillary voltage: 2000 V (positive ion mode) or
− 1500 V (negative ion mode); cone voltage: 30 V; ion source temper­
ature: 150 ◦ C; desolvent gas temperature: 500 ◦ C; backflush gas flow
rate: 50 L/h; desolventizing gas flow rate: 800 L/h. The injection volume
was 1 μL.
2.6.3. Data annotation and analysis
The raw data collected using Mass Lynx V4.2 is processed by Pro­
genesis QI software for peak extraction, peak alignment and other data
processing operations, based on the Progenesis QI software online
METLIN database and an in-house database (Biomarker Technologies
Co., LTD.) for identification, and at the same time, all of the theoretical
fragment identification and mass deviation are within 100 ppm. After
4
Food Chemistry 436 (2024) 137788
normalizing the original peak area information with the total peak area,
the follow-up analysis was performed. The detailed information on all
mentioned metabolites can be found in Table S4.
2.6.4. Quantitative of key precursors by UHPLC-MS/MS
7 organosulfur precursors and 8 key amino acids was performed by
external standardization method as described by Liu et al. (2019).
Standards were weighed (accurate to 0.01 mg) and dissolved in a 10 mL
volumetric flask with different ratios of methanol and water according
to different polarity of the compounds as standard stock solutions. Then
appropriate volume of standard stock solutions was mixed and diluted
with methanol/water (50/50, v/v) to a mass concentration of 10 mg/L
which was stored at − 20 ◦ C until use. Mixed standard solution was then
diluted to 1–1000 μg/L with 0.1 % formic acid aqueous solution. The
final working solution was prepared on the spot. The standardized
curves and methodological examination of quantitative were listed in
Table S9.
Samples (5.0 g) were ground liquid nitrogen and added into in a 50-
mL centrifuge tube with 20 mL aqueous solution containing 0.1 % for­
mic acid (pH < 3). The tube was vortexed for 1 min and subsequently
sonicated for 10 min at 25 ◦ C. The samples were centrifuged at 10,000
rpm for 10 min, and the supernatants were passed through an organic
microfilter (Nylon, 0.22 μm, Sartorius, Germany) before analysis. The
origin extracts of the samples were diluted 20-fold and 400-fold for
analysis. Samples were analyzed by an1290 Infinity II LC system coupled
to a G6465B Ultivo MS/MS system. Chromatographic separations were
conducted using an Xbridge C18 column (4.6 mm × 150 mm, i.d., 3.5
μm; Waters) at 40 ◦ C. The detail of gradient elution was listed in
Table S5. The injection volume was 5 μL. Mass spectrometry was using
electrospray ionization (ESI) in the positive ion mode. Quantification
was performed using the multiple reaction monitoring (MRM) mode to
monitor the precursor-product ion transition, and the corresponding
parameters were presented in Table S6.
2.7. RNA extraction and sequencing analysis
RNA extraction and sequencing analysis of TSB and TSM were per­
formed by Metware Co., Ltd, China (https://www.metware.cn/).
Following manufacturer’s recommendations, sequencing libraries were
generated using NEBNext®UltraTM RNA Library Prep Kit. Then PCR
was performed with Phusion High-Fidelity DNA polymerase, Universal
PCR primers and Index (X) Primer. Briefly, the purity, integrity and
concentration of the purified RNA were assessed using the Agilent Bio­
analyzer 2100 system. The library preparation was sequenced on an
Illumina platform and 150 bp paired-end reads were generated. The N
content and low-quality reads were removed to filter the original data by
fastpv0.19.3. The index was constructed using HISAT v2.1.0 and clean
reads were compared with the reference genome from the website (http
s://db.cngb.org/search/project/CNP0000958//). The gene alignment
and FPKM were calculated by feature Countsv1.6.2 /String Tiev1.3.4d.
The annotation by homology search against the following databases: Nr,
Pfam, KOG, COG, Swiss-Prot, KEGG, and GO. The detailed information
about the all genes mentioned in this work can be found in Table S7.
2.8. RT-qPCR analysis
A set of DEGs identified in this research were selected for qRT-PCR
verification. Gene-specific primers used in the analysis were synthe­
sized by Sangon Biotech (Shanghai, China) and listed in Supplementary
Table S8. Actin was used as a reference gene to normalize all gene
expression levels by the previous reports (Zhao et al., 2021). The 2-△△Ct
method was used to calculate relative expression levels of genes. Three
biological replicates and three technical replicates were assessed.
B. Zhang et al. Food Chemistry 436 (2024) 137788

Table 1
The composition and contents of the identified VOCs in TSB and TSM.
RIb Contents(μg/g) Abbreviationsd Identification CASf
RTa Volatile compounds HP- DB-5 TSB TSM Methods e
Innowax

6.97 Propanal 798.4 ndc 0.88 ± 1.42 ± 0.17 Ald-1 A; C 123–38-6

0.13
8.78 Allyl mercaptan 935.6 nd 32.78 ± 73.4 ± 7.55 Sul-1 A; C 870–23-5
1.6
9.87 (E)-1-(Methylthio)-1-propene 1002.4 nd nd 4.02 ± 1.03 Sul-2 A 42848–06-6
10.38 (Z)-1-(Methylthio)-1-propene 1045.3 nd 1.14 ± 1.44 ± 0.3 Sul-3 A 52195–40-1
0.09
10.44 α-Pinene 1050.3 797.1 3.39 ± nd Ter-1 A; B; C 80–56-8
0.28
11.57 Hexanal 1121.4 856.1 3.02 ± 11.54 ± Ald-2 A; B; C 66–25-1
0.42 1.05
12.02 (E)-2-Methyl-2-butenal 1139.3 nd 1.71 ± 0.3 nd Ald-3 A; C 497–03-0
12.25 2-Methyl-2-bornene 1148.5 871.1 nd 1.21 ± 0.05 Ter-2 B 72540–93-3
12.95 β-Myrcene 1176.3 nd 1.88 ± 0.91 ± 0.14 Ter-3 A; C 123–35-3
0.06
13.18 1- Propenyl 1-propyny sulfide 1185.5 876.6 0.5 ± 0.12 1.93 ± 0.22 Sul-4 A; B 89533–93-7
13.84 2,5-Dimethyl thiophene 1214.3 902.9 6.21 ± 6.99 ± 1.33 Sul-5 A; B 638–02-8
0.27
13.98 (Z)-Allyl(prop-1-en-1-yl)sulfane 1221.1 nd 0.73 ± 2.23 ± 0.12 Sul-6 A 104324–69-
0.14 8
14.10 β-Phellandrene 1226.9 nd 0.99 ± 0.1 nd Ter-4 A; C 555–10-2
14.17 Eucalyptol 1230.3 nd 0.91 ± nd Alc-1 A; C 470–82-6
0.08
14.22 (E)-Allyl(prop-1-en-1-yl)sulfane 1232.7 nd 1.63 ± 2.6 ± 0.4 Sul-7 A 104324–36-
0.38 9
14.45 2-Hexenal 1243.9 919.0 8.9 ± 0.59 5.89 ± 0.15 Ald-4 A; B; C 505–57-7
14.93 trans-β-Ocimene 1267.2 928.8 2.43 ± nd Ter-5 A; B; C 3779–61-1
0.11
15.17 3,4-Dimethyl thiophene 1278.9 932.2 78.54 ± 103.36 ± Sul-8 A; B 632–15-5
9.37 12.98
15.28 2,4-Dimethyl thiophene 1284.2 1016.1 nd 10.14 ± Sul-9 A; B 638–00-6
2.72
15.36 o-Cymene 1288.1 nd 0.32 ± nd Ter-6 A; C 527–84-4
0.03
15.62 1-Methyl-4-(1-methylethylidene) cyclohexene 1300.6 nd 0.56 ± nd Ter-7 A; C 586–62-9
0.04
16.73 (E)-1-Methyl-2-(prop-1-en-1-yl)disulfane 1348.3 1043.9 nd 4.67 ± 0.63 Sul-10 B; C 23838–19-9
17.03 6-Methyl-5-hepten-2-one 1361.2 nd 0.54 ± nd Ket-1 A; C 110–93-0
0.12
17.10 1-Hexanol 1364.3 nd 1.1 ± 0.05 nd Alc-2 A; C 111–27-3
17.41 (Z)-3-Hexen-1-ol 1377.6 nd 0.89 ± nd Alc-3 A; C 928–96-1
0.07
18.24 Nonanal 1412.1 nd 2.2 ± 0.42 nd Ald-5 A; C 124–19-6
18.51 α-Naginatene 1422.7 nd 4.48 ± nd Oth-1 A; C 15186–51-3
1.19
20.04 α-Cubebene 1482.6 1138.3 23.88 ± 4.01 ± 0.78 Ter-8 A; B; C 17699–14-8
1.65
20.66 Elixene 1506.8 nd 1.55 ± nd Ter-9 A; C 3242–08-8
0.15
21.04 Copaene 1520.4 1139.4 14.88 ± 3.53 ± 0.59 Ter-10 A; B; C 3856–25-5
3.51
21.87 (-)-β-Bourbonene 1551.0 nd 0.4 ± 0.04 0.64 ± 0.13 Ter-11 A; C 5208–59-3
21.94 β-Cubebene 1553.6 nd 5.87 ± nd Ter-12 A; C 13744–15-5
0.92
22.01 7-epi-Sesquithujene 1556.2 nd 3.1 ± 0.14 2.14 ± 0.44 Ter-13 A 159407–35-
9
22.07 2-Ethyl[1,3]dithiane 1558.4 1155.0 nd 3.26 ± 0.82 Sul-11 A; B 6007–23-4
22.82 (Z)-Prop-1-en-1-yl propanedithioate 1586.1 1160.0 32.63 ± 4.36 ± 0.5 Sul-12 A; B 67230–81-3
7.06
22.97 (E)-Prop-1-en-1-yl propanedithioate 1591.6 1184.4 45.5 ± 9.55 ± 1.5 Sul-13 A; B 67269–06-1
9.01
23.45 cis-Caryophyllene 1608.5 1216.0 18.75 ± 9.74 ± 1.7 Ter-14 A; B 13877–93-5
0.75
23.79 β-Elemene 1620.0 1290.1 63.14 ± 34.23 ± Ter-15 A; B; C 515–13-9
3.02 7.96
24.16 Caryophyllene 1632.5 1330.9 63.86 ± 29.78 ± Ter-16 A; B; C 87–44-5
1.18 4.89
24.30 β-Cedrene 1637.2 1339.3 1.33 ± 0.2 6.59 ± 1.37 Ter-17 B; C 546–28-1
24.44 β-Cyclocitral 1641.9 1343.8 2.6 ± 0.44 nd Ald-6 A; B; C 432–25-7
24.56 Aromadendrene 1646.0 1361.2 9.42 ± 12.61 ± Ter-18 A; B 489–39-4
0.43 1.71
24.66 Selina-5,11-diene 1649.4 1371.3 3.3 ± 0.54 nd Ter-19 A; B 52026–55-8
(continued on next page)

5
B. Zhang et al. Food Chemistry 436 (2024) 137788

Table 1 (continued )
RIb Contents(μg/g) Abbreviationsd Identification CASf
RTa Volatile compounds HP- DB-5 TSB TSM Methods e
Innowax

24.72 γ-Elemene 1651.4 1385.4 5.19 ± nd Ter-20 A; B 29873–99-2

0.24
25.29 cis-2-Mercapto-3,4-dimethyl-2,3-dihydrothiophene 1670.7 1400.6 175.5 ± 178.93 ± Sul-14 B; C -g
24.68 8.02
25.63 Alloaromadendrene 1682.1 1416.4 9.16 ± nd Ter-21 B; C 25246–27-9
0.25
25.85 4-Cyclohexylidene-3,3-diethyl-2-pentanone 1689.6 1421.6 nd 53.66 ± Ket-2 B 313253–65-
6.07 5
26.54 β-Chamigrene 1711.4 nd 40.93 ± nd Ter-22 A; C 18431–82-8
9.55
26.67 γ-Muurolene 1715.3 1425.7 9.14 ± nd Ter-23 A; B; C 30021–74-0
0.46
27.47 Ledene 1739.3 nd 9.61 ± nd Ter-24 A; C 21747–46-6
0.45
27.93 trans-2-Mercapto-3,4-dimethyl-2,3-dihydrothiophene 1753.1 1435.7 73.99 ± 170.88 ± Sul-15 B; C –
10.4 7.14
27.95 Eremophilene 1753.7 nd 11.54 ± nd Ter-25 A; C 10219–75-7
2.12
28.04 β-Selinene 1756.4 1442.1 59.11 ± 1.94 ± 0.46 Ter-26 A; B; C 17066–67-0
14.56
28.12 α-Selinene 1758.8 1457.3 57.67 ± 14.55 ± Ter-27 A; B; C 473–13-2
11.44 2.36
28.26 β-Dihydroagarofuran 1763.0 1467.3 nd 16.57 ± Oth-2 A; B 5956–09-2
1.16
28.34 Bicylogermacrene 1765.4 nd 11.39 ± 6.14 ± 0.43 Ter-28 A; C 24703–35-3
3.39
28.87 δ-Cadinene 1781.2 1473.1 39.93 ± 11 ± 1.82 Ter-29 A; B; C 483–76-1
8.57
29.58 γ-Cadinene 1802.5 1486.5 40.17 ± 15.15 ± Ter-30 A; B; C 39029–41-9
6.4 2.38
29.68 α-Maaliene 1805.5 nd 6.56 ± 8.35 ± 1.13 Ter-31 A 489–28-1
1.77
30.11 Cubenene 1818.4 nd 9.43 ± nd Ter-32 A; C 29837–12-5
2.26
30.24 10-Hydroxydecanoic acid, methyl ester 1822.3 1493.6 73.08 ± 48.05 ± 5.3 Ester B; C 2640–94-0
4.53
30.45 1,3-Di((E)-prop-1-en-1-yl)trisulfane 1828.6 1503.0 2.66 ± nd Sul-16 A; B 115321–81-
0.53 8
30.56 (Z)-1-(Prop-1-en-1-yl)-3-propyltrisulfane 1831.9 1510.3 2.39 ± 6.49 ± 0.98 Sul-17 A; B 23838–26-8
0.72
31.22 6-(3-Isopropenylcycloprop-1-enyl)-6-methylhept-3-en- 1851.7 1516.4 0.53 ± 1.42 ± 0.04 Ket-3 B –
2-one 0.06
31.46 trans-Calamenene 1858.9 1519.4 13.43 ± 7.27 ± 1.15 Ter-33 A; B 73209–42-4
2.96
32.57 cis-Eudesm-6-en-11-ol 1892.1 1539.4 5.9 ± 1.06 43.35 ± Alc-4 A; B 194607–96-
3.76 0
34.44 Calacorene 1948.2 1564.8 7.91 ± 3.71 ± 0.36 Ter-34 A; B; C 21391–99-1
2.23
34.76 (1S,3aR,4R,8R,8aS)-1-Isopropyl-3a-methyl-7- 1957.7 nd nd 1.45 ± 0.4 Oth-3 A; C 88395–47-5
methylenedecahydro-4,8-epoxyazulene
35.48 Shyobunol 1979.3 1574.5 1.37 ± nd Alc-5 B 35727–45-8
0.37
35.95 β-Calacorene 1993.4 nd 0.99 ± nd Ter-35 A 50277–34-4
0.12
36.36 β-Vatirenene 2005.7 1578.2 0.6 ± 0.09 nd Ter-36 A; B 27840–40-0
37.14 Caryophyllene oxide 2029.1 nd nd 1.2 ± 0.12 Oth-4 A; C 1139–30-6
39.51 Junenol 2100.1 1592.7 nd 9.54 ± 0.19 Alc-6 A; B 472–07-1
40.56 Methyl 1-(1-propenylthio)propyl disulfide, 2131.5 1622.3 6.26 ± 22.58 ± 0.6 Sul-18 A; B 126876–23-
0.86 1
41.10 β-Acorenol 2147.7 1626.1 1.09 ± 1.31 ± 0.17 Alc-7 B 28400–11-5
0.33
41.38 (-)-Spathulenol 2156.1 1640.8 0.9 ± 0.14 6.21 ± 1.02 Alc-8 B 77171–55-2
41.92 Eugenol 2172.3 nd 5.22 ± nd Oth-5 A; C 97–53-0
0.56
42.14 τ-Cadinol 2178.9 1715.9 5.56 ± 5.66 ± 0.95 Alc-9 A; B; C 5937–11-1
0.99
42.80 Mintsulfide 2198.7 1742.8 7.76 ± nd Sul-19 A; B 72445–42-2
0.95
43.24 τ-Muurolol 2211.8 nd 2.33 ± 2.69 ± 0.21 Alc-10 A; C 19912–62-0
0.34
44.75 α-Cadinol 2257.1 nd 4.8 ± 0.78 1.91 ± 0.49 Alc-11 A; C 481–34-5
45.46 trans-Geranylgeraniol 2278.4 1832.4 2.36 ± 0.2 13.1 ± 1.11 Alc-12 A; B 24034–73-9

6
B. Zhang et al. Food Chemistry 436 (2024) 137788

Note: a Retention time by HP-Innowax column; b Retention indices by Van Den Dool and Kratz; c nd: Not determined; d Abbreviations of VOCs in this study; e methods
(A) SI or/and RSI score was over 800; (B) SI or/and RSI score was over 700 compounds and was recognized by both columns of different polarities; (C) the difference
between calculated retention indices (RIc) and reference retention indices (RIf) were less than 30; f CAS number; g compound with no CAS number.

18 Ter
er
13 VSC
A 7 Alc D
3 Ald
1 Ket
1 Est

17 Ter 1 Ter
2 VSC 4 VSC
4 Alc 1 Alc
3 Ald 1 Ket
1 Ket 3 Oth
2 Oth

B Sul Ter Alc Ald Oth Ket Est

TSM 60.36 17.26

TSB 40.70 47.97

0% 20% 40% 60% 80% 100%

Relatively contents/%
C

Fig. 2. Volatile profiles of TSB and TSM. The Venn diagram of VOCs (A), the relative contents of each class (B), the total contents of each class (C) and the heatmap of
the shared VOCs (D).

2.9. Statistical analysis 3. Results and discussion

All experimental data in this work are expressed as mean ± standard
deviation (SD). The t-test was used to calculate the difference signifi­
cance p-value by SPSS 26.0 (IBM Corporation, USA). The PCA and OPLS-
DA analysis were performed by SIMCA 14.1. The cluster heatmap was
visualized by TBtools (version 1.082, China).
3.1. The overall aroma characteristics of TSB and TSM
E-nose was firstly used to obtain overall aroma characteristics in TSB
and TSM. Among the 10 metal oxide sensors, W1W, W2W, W5S and W1S
showed high responses in both TSB and TSM. The results indicated that
the high abundances of sulfur compounds, terpenes, nitrogen oxides and
methyl-branched VOCs may exist in both samples (Fig. 1C). Differently,
the responses of W1W and W2W were clearly stronger in TSM than in
TSB. The two sensors were both highly sensitive to sulfur compounds,

7
B. Zhang et al. Food Chemistry 436 (2024) 137788

Fig. 3. Score scatter plot of principal component analysis (PCA) (A) and orthogonal partial least squares-discriminant analysis (OPLS-DA) (B), the loading plot of all
identified VOCs in TSB and TSM OPLS-DA (C), the contents of differential VOCs screened by OPLS-DA and their variable important in projection (VIP) values (D).

8
B. Zhang et al. Food Chemistry 436 (2024) 137788

trans-

Fig. 4. Profiles of non-volatile intermediates and the genes encoding enzymes involved in the volatile sulfur compounds biosynthesis. Potential biosynthetic
pathways of volatile sulfur compounds in TSB and TSM (A) and heatmap of the FKPM values of each identified genes (B). The solid and dotted arrows represented the
one-step and multi-step reactions, respectively.

indicating the higher levels of VSCs in TSM. Compared with TSM, the
higher responsiveness of W1S, W2S and W3S existed in TSB, indicating
that methyl-branched VOCs, alcohols and long-chain alkenes were
abundant in TSB. According to the PCA plot (Fig. 1D), the accumulative
variance contribution rate of the first two principal components could
reach to 98.5 %, which demonstrated good ability in distinguishing
these two samples. In other words, E-nose could be used as an effective
tool to recognize the differences between the two similar TS-like aromas
of TSB and TSM by the overall odor attributes. However, the specific
components of the difference were still unclear.
3.2. Volatile metabolites analysis of TSB and TSM
3.2.1. Overview of VOC profiles in TSB and TSM
To ensure the VOC identification accuracy in TSB and TSM samples,
the samples were detected by HS-SPME-GC/MS analysis with polar and
non-polar columns, respectively. As a result of numerous polar VOCs
being observed in TSB and TSM samples, the quantitative analysis was
based on the HP-Innowax column in this work (Wang et al., 2023a; Yang
et al., 2019). As shown in Table 1, a total of 82 VOCs were identified by
the methods described in section 2.4.3, which contained 36 terpenes, 19
VSCs, 12 alcohols, 6 aldehydes, 3 ketones, 1 ester and 5 other com­
pounds. Thereinto, only 43 VOCs were found in both samples (Fig. 2A),
which accounted for 87 % and 89 % of the total VOC contents in TSB and
TSM, respectively. As a perfect substitute for TSB, the similar odor was
often reported in TSM (Li et al., 2013). However, the obvious differences
of VOC profiles were also evident in TSM and TSB samples. For example,
the higher contents of the total VSCs were found in TSM (606.82 μg/g)
than that of TSB (468.24 μg/g) (Fig. 2B-C). Conversely, the total con­
tents of terpenes in TSB (551.81 μg/g) were over three times than that of
TSM (173.50 μg/g) (Fig. 2B-C). The similar results were also observed in
the clustering analysis of heat map (Fig. 2D). To reconcile the irritating
odor of VSCs, in fact, the soft odors of terpenes were commonly recog­
nized as the important VOCs in TSB. Compared with TSB, therefore, how
to balance the irritating odor of the higher VSCs contents with the lower
terpene contents in TSM is still unclear.
3.2.2. Principal component and multivariate analysis of differential VOCs
As one of the most widely used multivariate analysis methods, PCA
and OPLS-DA were efficient ways to explore the specific characteristics
of VOC profiles (Yang et al., 2019). As seen in Fig. 3A-B, the samples of
TSB and TSM could be distinguished obviously in both PCA and OPLS-
DA analysis. The model validation was shown to be reliable with all
intercepts of the Q2 regression line below zero. According to the OPLS-
DA loading plot (Fig. 3C), it was evident that several VOCs showed the
longer distances from the origin point, such as terpenes (e.g., Ter-22,
Ter-26, Ter-27, Ter-29), VSCs (e.g., Sul-1, Sul-14, Sul-15), alcohols (e.
g., Alc-4), ketones (e.g., Ket-2), and so on. To identify the contribution of
each VOC in distinguishing TSB and TSM samples, the variable impor­
tance of projection (VIP) of all VOCs was calculated (Segers et al., 2019).
By using VIP score＞1.0 and p＜0.05 as the screening conditions, 20
VOCs were successfully identified, containing 10 terpenes, 5 VSCs, 2
alcohols, 1 ketone, 1 ester and 1 other compound (Fig. 3D).
Among the identified VOCs, Sul-15 (trans-2-mercapto-3,4-dimethyl-
2,3-dihydrothiophene) showed the highest VIP score (VIP = 3.04). The
content of Sul-15 was more than twice as high in TSM as in TSB. It was
noteworthy that Sul-14, an isomer of Sul-15, showed a significant far
distance from the origin point in Fig. 3C, but it displayed the low VIP
score. However, the high contents of Sul-14 were observed in TSB
(175.50 μg/g) and TSM (178.93 μg/g). In fact, Sul-14 and Sul-15 were
commonly recognized as the typical odors of T. sinensis in several studies
(Liu et al., 2013; Wang et al., 2020; Yang et al., 2019; Zhai & Granvogl,

9
B. Zhang et al.
2020a). In this work, the total contents of these two compounds could
reach 21.69 % and 34.80 % of the total VOCs contents in TSB and TSM,
respectively. It was suggested that 2-mercapto-3,4-dimethyl-2,3-dihy­
drothiophene contributed extremely high to the overall aroma of these
two samples. Nevertheless, previous literatures reported that Sul-14 and
Sul-15 could not be biosynthesized in T. sinensis, both of which were
mainly formed by dithio-Claisen rearrangement reaction from bis-(1-
propenyl) disulfides during high-temperature conditions (e.g., GC
injector) (Yang et al., 2019). Specifically, bis-(1-propenyl) disulfides can
be formed from the polymerization reaction of 1-propenyl-1-thiol (an
unstable isomer of Sul-1, allyl mercaptan, VIP = 1.94) (Yang et al.,
2019). The VSCs containing a 1-propenyl sulfide group were also iden­
tified as the key precursors in the aroma formation of T. sinensis. In fact,
the other VSCs in VIP analysis can be also synthesized by the sponta­
neous of 1-propenyl-1-thiol or 1-propenylsulfenic acid, such as (Z)-prop-
1-en-1-yl propanedithioate (Sul-12, VIP = 1.61), (E)-prop-1-en-1-yl
propanedithioate (Sul-13, VIP = 1.81), methyl 1-(1-propenylthio) pro­
pyl disulfide (Sul-18, VIP = 1.25). Therefore, 1-propenyl-1-thiol and its
isomer (Sul-1) might be the crucial precursors of the distinctive sulfur-
flavor formation in T. sinensis.
Apart from these above VSCs, a large number of terpenes also dis­
played the high VIP scores, including Ter-8, Ter-10, Ter-15, Ter-16, Ter-
22, Ter-25, Ter-26, Ter-27, Ter-29 and Ter-30. Specially, all of these
VOCs were sesquiterpenes and thereinto, several terpenes (e.g., Ter-15,
Ter-16 and Ter-26) had been reported as the important VOCs in
T. sinensis before (Wang et al., 2020). Compared with TSM, all of these
terpenes showed higher contents in TSB (Table 1 and Fig. 3D). For
example, the content of Ter-26 (β-selinene, 59.11 μg/g) was 30.47-fold
higher than that of TSM (1.94 μg/g). The contents of Ter-15 and Ter-16
in TSB were also over 1.8-folds than that of TSM (Table 1). As we all
know, terpenes are commonly used as a signal in stressful environments
(e.g., pests and disease) (Brosset & Blande, 2022). By comparison, the
growth of TSB would face complex environmental stresses than TSM.
Hence, biotic or abiotic stresses might be the potential reasons for the
higher terpene contents in TSB. As the main VOCs in reconciling the
irritating odor of VSCs, however, the significant higher contents of VSCs
were observed in TSM (606.85 μg/g) than that of TSB (468.24 μg/g)
(Fig. 2C). Under the low contents of terpenes, how to balance the irri­
tating odor of the abundant VSCs in TSM is an important question. In
addition to the terpenes, another interesting finding was that the con­
tents of alcohols and ketones exhibited higher levels in TSM than in TSB.
For example, Alc-4 (cis-eudesm-6-en-11-ol, VIP = 1.63), Alc-12 (trans-
geranylgeraniol, VIP = 1.63), Ket-2 (4-cyclohexylidene-3,3-diethyl-2-
pentanone, VIP = 2.27) and Oth-2 (β- dihydroagarofuran, VIP = 1.26)
could not only exhibit the high VIP scores, but also own the high con­
tents in TSM. Therefore, these VOCs might play crucial roles in
balancing the irritating odor of VSCs in TSM.
3.3. Metabolic changes of the main VOCs biosynthetic pathways
3.3.1. Analysis of non-volatile metabolites and genes related to VSCs
biosynthesis
To explore the potential differences in aroma formation, the intra­
cellular metabolism was further compared in TSB and TSM by UPLC-
MS/MS and RNA sequencing analysis. According to the previous re­
ports by our group, the potential VSCs biosynthetic pathways had been
speculated in TSB (Wang et al., 2023b). As shown in Fig. 4, the gene
expression levels and non-volatile intermediate metabolites involved in
VSCs biosynthesis were detected and analyzed in TSB and TSM. By
KEGG enrichment analysis, 24 non-volatile metabolites (Table S4 and
Fig. 4A) and 88 genes (Table S7 and Fig. 4B) were screened out and
enriched into 6 metabolic pathways.
In the sulfate assimilation pathway, most genes (except for ATP
sulfurylase (APS)) showed the higher expression level in TSB than in
TSM, such as adenylyl-sulfate kinase (PAPS), sulfite reductase (SIR and
CPP), and cystathionine gamma-synthase (MET) (Fig. 4B). It indicated
10
Food Chemistry 436 (2024) 137788
that a large amount of sulfate from the environment had been fluxed into
the plant tissues of TSB. While TSM was hydroponically grown, the
source of sulfur for which was mainly obtained by the conversion of
energy substances stored in seed, such as sulfur-containing amino acids.
In the H2S assimilation pathways, all identified genes encoding MET
(cystathionine gamma-synthase) exhibited comparably higher expres­
sion levels in TSB. Conversely, the high expression levels of the genes
encoding cysteine synthase (CYSK) were observed in TSM. As two key
H2S assimilation pathways, notably, the levels of the relative substrates
(i.e., O-acetyl homoserine, O-acetyl serine) catalyzed by these two en­
zymes also exhibited the obvious differences in TSM and TSB. The high
expression of CYSK might lead to excessive consumption of O-acetyl
serine, leading to a lower level of O-acetyl serine in TSM (Table S4).
These results indicated that more H2S was incorporated into cysteine
synthesis by serine transformation than through aspartate catabolism in
TSM. But for TSB, the opposite tendency may exist.
During aspartate degradation, homocysteine can also be converted
back to methionine through the methionine-homocysteine cycle (Li,
2015). The differential expression of these genes may impact the
intracellular sulfur metabolism in TSB and TSM. For example, the genes
encoding S-adenosylmethionine synthetase (METK) and S-adenosylme­
thionine decarboxylase (SAMD) showed higher expression in TSM than
in TSB. Especially, the highest FPKM values of SAMD could reach to
2699.81 and 2.92-folds in TSM compared to TSB. As an active methyl
donor for numerous reactions in plants (Amir, 2010), more S-adenosyl-L-
methionine may be consumed by the high expression level of SAMD.
Accordingly, the high relative abundances of S-adenosyl-methionin­
amine and 1-aminocyclopropane-1-carboxylate were also observed in
TSM (Table S4). On the contrary, the lower expression levels of genes
encoding methyltransferase (DNMT and YRRT) were found in TSM than
in TSB. These results indicated that more metabolic flux flowed out of
the methionine-homocysteine cycle in TSM. That is the potential reason
that the genes encoding cystathionine beta-synthase (MCCA) displayed
the high expression levels in TSB than that of TSM.
As the key sulfur sources of VSCs biosynthesis, cysteine can be firstly
converted to glutathione and further degraded into different VSCs by S-
oxygenation and deglutamylation processes (Yamaguchi & Kumagai,
2020; Yoshimoto & Saito, 2019). Notably, the relative abundance of S-
(trans-L-propenyl)-L-cysteine was significantly higher than that of S-
methyl-L-cysteine in both TSB and TSM (Table S4). According to the
structural analysis of VSCs in the blue box of Fig. 4A, a branched chain
containing a 1-propenyl sulfide group was repeatedly observed. There­
fore, we speculated that S-(trans-L-propenyl)-L-cysteine might be the
main precursor of VSCs biosynthesis in T. sinensis.
To validate the accuracy of this hypothesis, we conducted additional
quantification of crucial sulfur-containing precursors and amino acids in
the metabolic pathway using UHPLC-MS/MS (Table S9). The results
showed that the abundance of S-(trans-L-propenyl)-L-cysteine (412.38
and 99.86 μg/g in TSM and TSB) was significantly higher than that of S-
methyl-L-cysteine (0.27 and 0.15 μg/g in TSM and TSB). This further
confirms our speculation mentioned above and supports the existence of
the similar metabolic pathways of VSCs in T. sinensis with that of Allium
plants (Wang et al., 2023b). It was worth noting that S-(trans-L-pro­
penyl)-L-cysteine, S-methyl-L-cysteine and γ-glutamyl-S-methyl-L-
cysteine were first identified as important precursors of VSCs in
T. sinensis. At the same time, the differences in the content of key amino
acids in TSB and TSM exhibited consistent patterns with previous rela­
tive quantitative results. Additionally, we also supplemented the con­
tents of undetected cysteine and glycine before, which were both higher
in TSM (Table S9). Strangely, we did not detect any corresponding S-alk
(en)ylcysteine sulfoxides (ACSOs) although the standard reagents were
used (Table S9). This could be attributed to the complex metabolic
changes during storage or the need for further optimization of the
method.
While the VSCs biosynthesis pattern was similar in both TSM and
TSB, the obvious differences in gene expression and metabolite levels
B. Zhang et al. Food Chemistry 436 (2024) 137788

Fig. 5. Profiles of non-volatile intermediates and the genes encoding enzymes involved in the terpenoids biosynthesis. Biosynthetic pathways of terpenoids in TSB
and TSM (A) and heatmap of the FKPM values of each identified gene (B).

11
B. Zhang et al.
were also been noted in the aforementioned metabolic pathways. For
example, most of these identified genes displayed the higher expression
levels in TSM than that of TSB, such as glutamate-cysteine ligase (GCLC),
glutathione synthase (GSS), glutamate carboxypeptidase (CPG), γ-glu­
tamyltransferase (GGT), flavin-containing monooxygenase (FMO-1,2).
Consistently, main precursors including γ-glutamyl-S-(trans-L-propenyl)-
L-cysteine and S-(trans-L-propenyl)-L-cysteine were found to be more
abundant in TSM. These results suggested that the biosynthesis ability of
VSCs was stronger in TSM than TSB. Interestingly, ACSOs could further
transform into some VSCs (e.g., 1-propenyl sulfenic acid) by alliinase
(ALL) or by spontaneous reactions (Block, 2009; Block et al., 2018). A
possible reason could be speculated that ACSOs was not the direct pre­
cursor of VSCs biosynthesis in T. sinensis, which might be firstly trans­
formed into other intermediate metabolites (e.g., 1-propenyl sulfenic
acid) and then synthesized the relative VSCs (Wang et al., 2023b;
Yoshimoto & Saito, 2019). The presence of lachrymatory-factor syn­
thase (LFS) catalyzed the formation of propanethial S-oxide from 1-pro­
penyl sulfenic acid. From this perspective, the high expression of LFS in
TSB would consume a large amount of 1-propenyl sulfenic acid. It would
further decrease the precursor sources of VSCs biosynthesis in TSB.
Compared with TSM, therefore, the lower contents of the total VSCs
were found in TSB.
3.3.2. Analysis of metabolites and genes related to terpenoids biosynthesis
In plants, terpenoid biosynthesis had been identified clearly,
including the methylerythritol phosphate (MEP) and mevalonic acid
(MVA) pathways (Brosset & Blande, 2022; Dong et al., 2016; Huang
et al., 2012; Zhang et al., 2021). Thereinto, monoterpenes and sesqui­
terpenes are usually synthesized through the MEP (in plastid) and MVA
(in cytosol) pathways, respectively (Morris et al., 2011; Zhang at al.,
2009). The results of non-volatile metabolomics and transcriptomics
analysis showed that only 4 intermediate metabolites and 45 genes
screened out were relevant to the two pathways (Fig. 5A-B, Table S4 and
Table S7).
Among the identified terpenoids, notably, sesquiterpenes showed the
highest numbers and the relative abundances in TSB and TSM (Table 1).
In fact, the biosynthesis of one sesquiterpene requires two molecules of
isopentenyl diphosphate (IPP) and one molecule of dimethylallyl
diphosphate (DMAPP) (Morris et al., 2011). The high demands of IPP
would be the key limiting factor in the formation of sesquiterpenes.
Therefore, we speculated that isopentenyl diphosphate isomerase (IDI),
which regulates the IPP/DMAPP pool and the flux in the terpene
biosynthesis pathways, and mevalonate diphosphate decarboxylase
(MPDC), the last step synthesizing IPP in MVA pathway were crucial
enzymes in the terpenoid biosynthesis of T. sinensis. The higher
expression levels of these genes were consistent with the results of richer
terpenes in TSB. Compared with TSM, the expression levels of mevalo­
nate kinase (MVK), phosphomevalonate kinase (PMVK) exhibited higher
in TSB. Similarly, most of genes had also displayed the high expression
levels in MEP pathway in TSB samples, such as methyl-D-erythritol 4-
phosphate cytidylyltransferase (IspD), 4-diphosphocytidyl-2-C-methyl-
D-erythritol kinase (IspE), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate
synthase (IspF), (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate syn­
thase (GCPE), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase
(IspH). Among all identified four intermediate metabolites, both
mevalonate-5P and mevalonate-3P displayed the high relative abun­
dances in TSB samples (Table S4), which was accordant with tran­
scriptomics results.
Different with TSB, it was clear to see that only these genes encoding
the first two steps in both MVA and MEP pathways exhibited the higher
expression levels in TSM, including hydroxymethylglutaryl-CoA syn­
thase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), 1-
deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-
phosphate reductoisomerase (DXR). During the germination of seeds, in
fact, the different types of carbohydrates (e.g., starch and fatty acids)
stored in seeds would be hydrolyzed and transformed to maintain the
12
Food Chemistry 436 (2024) 137788
cell growth. For example, acetyl-CoA could be formed during the
degradation of fatty acids. Starch could be hydrolyzed into mono­
saccharide and further participate in intracellular metabolism through
the glycolysis pathway. As extremely important intracellular metabo­
lites, therefore, some of these compounds (e.g., acetyl-CoA, D-glyceral­
dehyde 3-phosphate) would exhibit the relatively higher levels in TSM.
In other words, the higher levels of these compounds were primarily
used for the cell growth rather than terpenoid biosynthesis. That is why
lower terpenoid abundances were found in TSM than in TSB.
3.4. Gene expression validation by RT-qPCR
To verify the accuracy of transcriptome data, qRT-PCR analysis was
performed on several genes related to the VOCs biosynthesis in
T. sinensis (Fig. S1A). Further, qRT-PCR results of all genes showed the
same expression trends in expression levels noted via RNA-seq
(Fig. S1B), providing strong evidence for the credibility and reliability
of this study.
4. Conclusion
In summary, this study investigated the aroma characteristics and
formation mechanism of TSB and TSM. Similar but distinct aroma
characteristics were identified in TSB and TSM. VSCs and terpenes were
the main VOCs and crucial biomarkers of TSB and TSM. Differences in
the predominant source of cysteine synthesis and the availability of key
precursor substances may affect the VSCs accumulations in T. sinensis. S-
(trans-L-propenyl)-L-cysteine might be the main precursor of VSC
biosynthesis compared to S-methyl-L-cysteine, although both of them
were identified for the first time as key non-volatile precursors for VSC
biosynthesis in T. sinensis. The transcriptome analysis revealed that high
expression of genes in MVA and MEP downstream caused more terpenes
synthesis in TSB. These findings provide experimental data and valuable
insights into the characteristic aroma formation and regulation in
T. sinensis.
CRediT authorship contribution statement
Beibei Zhang: Conceptualization, Methodology, Supervision,
Formal analysis, Writing – original draft, Writing – review & editing.
Lifang Hao: Methodology. Jing Zhang: Methodology. Jinze Feng: .
Cheng Wang: Investigation, Data curation, Writing – review & editing.
Jingfang Zhang: Validation, Writing – review & editing, Funding
acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
This work was supported by National Promotion Project of Forestry
and Grassland Science and Technology Achievements (2023133140),
Science Technology Innovation Project of Shaanxi Academy of Forestry
(SXLK2021-02) and Scientific Research Plan Projects of Shaanxi Edu­
cation Department (22JKO533).
We thank to Doctor Liu Pingxiang from Shandong Academy of
Agricultural Sciences for her assistant and providing the standards. And
we thank Horticulture Science Research Center at College of Horticul­
ture, NWAFU and Xi’an Qingzhihe Agriculture and Forestry Technology
B. Zhang et al.
Ltd. for their technical support in this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2023.137788.
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