healthmedicinet

Changes in volatile metabolic profile after MeJA treatment

According to the results of two-dimension GC-TOF/MS, the volatile metabolites in MeJA-treated
tea leaves changed significantly. We divided the identified metabolites into three
groups: C
₆
–C
₉
, C
₁₀
–C
₃₀
, and the others mainly including some acids. We could clearly figure out that major
of content of the flavor volatiles changed significantly after 12 h and 24 h treatment.
Most of the volatile compounds in the C
₆
–C
₉
category were increased in MeJA_12h treated tea leaves than the ck_12h. 2-Hexenal
is important for tea aroma, and responded indirectly to abiotic stress; according
to Table 1, the 2-Hexenal content increased to 9.62 ?g/g which implied massive biosynthesis
of this small-molecular-volatile metabolite after MeJA treatment. Similar results
were observed in the C
₁₀
–C
₃₀
category. Linalool, geraniol, methyl salicylate and phenylethyl alcohol are considered
the floral aroma contributors in brewed tea. The content of these four volatile metabolites
increased 1.91, 4.4, 0.91 and 9.25 ?g/g in MeJA_12h treated tea leaves, respectively,
and increased 1.65, 3.58, 5.54 and 5.09 ?g/g in MeJA_24 h treated tea leaves, respectively.
These results showed a prolonged increase in these four metabolites during MeJA treatment.

Table 1. Volatile compounds and some aroma-relative acid precursors in MeJA-treated tea leaves

Much more interestingly, we found most of the acid precursors, such as Salicylic acid,
trans-Cinnamic acid, Homovanillic acid, trans p-Coumaric acid, the majority of which
are related to volatile biosynthesis pathways, decreased in MeJA-treated 24 h tea
leaves. We hypothesized that these acids were consumed as precursors in synthesis
volatiles faster than they could be biosynthesized, resulting increased volatiles
contents and decreased contents of acid precursors.

Also the content of Octadecanoic acid was decreased significantly after methyl jasmonate
treatment, it is the intermediates of the ?-linolenic acid metabolism pathway which
finally synthesis massive JA and methyl jasmonate (Table 1).

Illumina sequencing and data analysis

RNA sequencing of the eight samples produced more than 27 million 100 bp paired-end
reads, with an average of 3 million reads for each sample. Cleaning and quality checks
were carried out on the raw data. More than 18 million trimmed reads were obtained
with useful data percentage ranging from 58.96 % to 72.23 %, and the average length
of each read was 195 bp (Additional file 1: Table S1). Compared with the reads generated by the formal platforms, the longer
length of Illumina Miseq sequencing reads aided the accuracy of the subsequent de
novo assembly, despite the lack of an available reference genome for tea. The de novo
assembly was performed using Trinity (http://trinityrnaseq.github.io/). All the short reads were assembled to generate 625,574 contigs with a mean size
of 290.15 bp and an N50 of 382 bp; 11.13 % reads of the samples were greater than
500 bp. Further assembly of the contigs generated components that were used to construct
a de Bruijn graph. Finally, optimizing the de Bruijn graph permitted us to build 320573
transcripts with average size of 796 bp and an N50 of 1392 bp (Table 2). All the transcripts were then BLAST searched against Arabidopsis database. For those sequences with no BLAST hits (non-BLASTable transcripts), we
searched them against the NCBI non-redundant (nr) database, using the BLASTx program
with an E-value threshold of 1E-5. To distinguish redundant sequences from homologous
sequences, unigenes were used in this study to minimize redundancy: each unique sequence
was assigned a unigene ID according to the accession number of the best-hit homolog
in the nr database. 50732 unigenes were obtained, with an average length of 1151 bp
(Table 2). The size distribution of contigs, transcripts and unigenes was compiled (Additional
file 2: Figure S1).

Table 2. Statistical summary of cDNA sequences of tea generated by Illumina Miseq platform

EggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) is a database
providing orthologous groups for 943 bacteria, 69 archaea and 121 eukaryotes. According
to previous studies, the proteins could be divided into 25 functional categories 24]. Out of 45745 unigenes with significant identity with nr database in this study,
40245 could be classified into 26 eggNOG categories (Additional file 3: Figure S2). The categories “function unknown” (8513, 21.15 %) and “general function
prediction only” (7383, 18.35 %) were the two largest functional groups among the
eggNOG categories. The high percentage of unigenes classified into “general function
prediction only” was similar to transcriptome studies of other species 25]–28]. The assignment of so many differential expressed unigenes to the unknown function
group suggested the presence of as yet unknown mechanisms of secondary metabolism
changes during the MeJA treatment of tea leaves. The next most abundant groups were
“Signal transduction mechanisms” (3330, 8.27 %), “Posttranslational modification”,
“protein turnover”, “chaperones” (3259, 8.10 %), “Translation”, “ribosomal structure
and biogenesis” (1964, 4.88 %), “Transcription” (1847, 4.59 %), whereas the groups
involving “cell motility” and “extracellular structures” consisted of a total of 80
unigenes (0.2 %), representing the smallest eggNOG classifications, excepting for
two undetermined unigenes. Notably, 1734 unigenes (4.31 %) and 1312 unigenes (3.05 %)
were classified into the carbohydrate metabolism and secondary metabolite biosynthesis
groups, respectively, including volatile compounds biosynthesis.

Differentially expressed gene analysis

To identify DEGs among MeJA-treated tea samples, we compared them with each other
and identified unigenes that were at least 2-fold up- or down regulated between the
two samples, with p-value less than 0.05. Then, hierarchical clustering was used to gain a global view
of DEGs (Fig. 3). The DEGs analysis of the MeJA_12h treated samples was similar to the MeJA_24h treated
samples. In total, 19245, 18614DEGs were identified in the MeJA_12h, MeJA_24h samples,
respectively (Fig. 1). These two are much more different from the MeJA-untreated ones. Thus, it was clear
that MeJA has a significant impact on the transcriptome of tea leaves. However, it
also could be deduced from the heat map that the MeJA_48 h samples were much more
special. It was different from the others, comparing with others, 11890 DEGs were
identified in the MeJA_48 h samples (Fig. 1), and the GO categories for the up- and down-regulated DEGs are shown separately
for the three main terms.

Fig. 1. Cluster of differentially expressed unigenes during MeJA treatment. Expression changes
and cluster analysis of 10,765 genes that were differentially expressed between any
two of four samples. Each row represents a differentially expressed gene, while each
column represents a sample. Changes in expression levels are shown in color scales
with saturation at 2.0-fold changes. Green and red color gradients indicate a decrease
and increase in transcript abundance, respectively

It was supposed that various genes were greatly affected within 48 h by MeJA treatment.
However, most of DEGs in 12, 24 h-MeJA samples are absolutely not the same as in 48 h-MeJA
and CK samples. Mostly, the expression of DEGs was improved within 24 h, then down-regulated.
We also know about that the MeJA treatment was much similar to herbivorous attack
that finally leading to mass consumption of plant its own. Expression of Genes, proteins
and content of metabolomics were firstly improved, then be consumed, and to the last,
recovered to the normal level.

The KEGG (Kyoto Encyclopedia of Genes and Genomes) is a database linking genomic information
with higher order functional information by collecting manually drawn pathway maps
representing current knowledge on cellular processes and standardized gene annotations.
To gain an overview of tea metabolic pathways that are modulated by MeJA, DEGs were
analyzed according to the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp). The analysis revealed a total of 45 KEGG pathways containing 20404 assigned unigenes
(Additional file 4: Table S2). The pathways with the largest numbers of assigned unigenes were “metabolism”,
“human disease” and “genetic information processing”. Furthermore, we performed KEGG
enrichment analysis of the DEGs identified in the MeJA_12h, MeJA_24h and MeJA_48h
samples compared with the MeJA-untreated samples respectively, and picked out 1406,
1443, 1695 DEGs which important in volatile related pathways (Fig. 2).

Fig. 2. Gene Ontology enrichment assigned to tea unigenes. GO categories of biological process,
cellular component and molecular function for the transcriptome of AR. Histogram presentation
of the gene ontology classification. The results are summarized in the three main
GO categories: biological process, cellular component and molecular function. a. Gene Ontology classification of 12 h methyl jasmonate-induced tea leaves; b. Gene Ontology classification of 24 h methyl jasmonate-induced tea leaves; c. Gene Ontology classification of 48 h methyl jasmonate-induced tea leaves. Note:
red line represents the p value?=?0.05

A list of secondary metabolic pathways represented by the unigenes is provided in
Table 3. Interestingly, among the secondary metabolic processes, terpenoids and phenylpropanoid
pathways were the most represented (Additional file 4: Table S2; Table 1). Strangely, these volatile secondary metabolite types were rarely reported to accumulate
at high levels in tea leaves after MeJA treatment; obviously, the DEGs involved in
the biosynthesis of these metabolites were not clearly identified.

Table 3. KEGG pathway analysis of the MeJA-responsive differential expressed unigenes

JA responsive pathways in MeJA-induced tea leaves

Interestingly, six DEGs were closely associated with the ?-linolenic acid metabolism
that finally leads to JA biosynthesis (Table 3; Fig. 3). The JA signaling pathway is the most important signal-transduction pathway in response
to predation and pathogen attack, acting as a “master switch” 7], 29]–31]. It may play a central role to trigger expression of those DEGs encoding lipoxygenase
(EC:1.13.11.12), acetyl-CoA acyltransferase 1 (EC: 2.3.1.16), two kinds of oxidase
(EC:1.3.3.6; EC:5.3.99.6) and jasmonate O-methyltransferase (EC:2.1.1.141). Previous
reports suggested that genes could perceive and respond to local and systemic signals
generated by external stimuli, including MeJA itself 32]–36]. During exogenous MeJA treatment, the expression level of jasmonate O-methyltransferase,
which catalyzes directly the substrates of (?)-JA biosynthesis, was upregulated by
7.52-fold compared with the control (Additional file 2: Figure S1, Additional file 5: Figure S3; Table 3). Free-acid JA might not be able to move across the cellular membrane without a carrier
because of its acidic nature; nonetheless, MeJA could diffuse to distal parts of plant
via the vapor phase or by intercellular migration 37], 38]. It is possible for exogenous MeJA to transfer into tea leaves, where it triggers
a series of fatty acid pathways resulting in biosynthesis of more JA and JA-conjuncts.
Finally, the JA-conjuncts may trig the whole plant’s JA pathway 39]–42].

Fig. 3. KEGG enrichment assigned to tea unigenes. a. KEGG enrichmen of 12 h methyl jasmonate-induced tea leaves; b. KEGG enrichmen of 24 h methyl jasmonate-induced tea leaves; c. KEGG enrichmen of 48 h methyl jasmonate-induced tea leaves. Note: red line represents
the p value?=?0.05

Responses of biosynthetic pathways of the flavor volatile compounds to MeJA

MeJA affects Fatty acid metabolism pathways

Jasmonic acid and its volatile methyl ester act as phytohormones, and are involved
in plant responses to stress and developmental processes. During MeJA treatment, the
fatty acid pathways are the first to respond, producing low molecular volatiles. At
least 13 enzymes are involved in the biosynthetic pathway leading to volatiles formation,
including lipoxygenase (LOX) (EC:1.13.11.58), acetyl-CoA acyltransferase 1 (ACAA1)
(EC:2.3.1.16), allene oxide cyclase (EC:5.3.99.6) and acyl-CoA oxidase (EC:1.3.3.6)
(Additional file 2: Figure S1).

In plants, fatty acids are stored as triacylglycerides; therefore, enzymatic oxidative
degradation of lipids is preceded by the action of acyl hydrolase, liberating the
free fatty acids from acylglycerols. Saturated and unsaturated volatile C
₆
and C
₉
aldehydes and alcohols are important contributors to the characteristic aromas of
tea, which are described as a “fresh green” odor. The short-chain aldehydes and alcohols
are mostly produced by plants in response to external stress and play an important
role in the plants defense strategies (Additional file 3: Figure S2A) 43]–47]. Quantitatively and qualitatively, the majority of plant volatiles originate from
saturated and unsaturated fatty acids. In tea plants, we identified many fatty acid-derived
straight-chain alcohols, aldehydes, ketones, acids, esters and lactones, which are
formed by three basic processes: ? -oxidation, ?-oxidation and the lipoxygenation.
According to Table 1, C
₆
-C
₉
volatiles: 2-ethoxy-Butane, 1-ethoxy-Butane, Cyclohexane, 1-ethoxy-Pentane, 2-methyl-Decane,
and 2,2-dimethyl-Propanal, increased immediately in MeJA-induced tea leaves. In addition,
large amounts of volatiles such as: 2-ethyl-1-Hexanol, 2-methyl-Decane, Acetaldehyde,
2,4-dimethyl-Heptane, 4-methyl-Octane, 1-methoxy-3-methyl-Butane, were synthesized
compared with MeJA-untreated tea leaves. The results shown in row d of Table 1 suggested that these volatiles were released quickly into the external environment
in response to recognition of exogenous threat. In particular, 2-Hexenal is a representative
volatile compound synthesized by fatty acid pathways, compared with the control, after
12 h of MeJA treatment the 2-Hexenal content had increased massively; subsequently,
it recovered to a normal level after 24 h of treatment. Interestingly, during the
procedure, 2-Hexenal was released quickly from tea leaves, suggesting that it had
an important impact on abiotic stress.

Taking these results together, in tea leaves subjected to the abiotic stress if MeJA
treatment, the JA pathway stimulation upregulates the fatty acid pathways, resulting
in rapid changes to the C
₆
–C
₉
volatiles profile.

MeJA affects terpenoids biosynthesis pathways

The most diverse family of natural products is the terpenoids, with over 40,000 different
structures. Various plants produce terpenoids, including volatile ones and non-volatile
ones. The volatile terpenoids (hemiterpenoids[C
₅
], monoterpenoids[C
₁₀
], sesquiterpenoids[C
₁₅
] and some diterpenoids[C
₂₀
]) are important in interactions between plants and insect herbivores, and are implicated
in exogenous elicitor-induced general defense or stress responses (Figs. 2a and 4) 48]–52]. Despite their diversity, all terpenoids are derived from the common building unit
isopentenyl diphosphate (IDP) and its isomer, dimethylallyl diphosphate (DMADP). Generally
speaking, the two 5C building blocks (DMADP and IDP) are formed via two independent
pathways: the mevalonic acid (MEV) pathway and the 2C-methyl-D-erythritol-4-phosphate
(MEP) pathway. IDP and DMADP derived from the cytosolic MEV pathway could serve as
precursors for the biosynthesis of the sesquiterpenes (C
₁₅
) and triterpenes (C
₃₀
), whereas the plastidial MEP pathway provides precursors for the biosynthesis of
the monoterpenes (C
₁₀
), diterpenes (C
₂₀
), and tetraterpenes (C
₄₀
) 53]–55].

Fig. 4. Biology response to of time-dependent methyl jasmonate treatment in tea leaves. a. exogenous methyl jasmonate could lead to a rapid, within minutes, oxidative burst
and release of free fatty acids and further cascade of events includes activation
of defense gene expression that leads to synthesis of a variety of volatile isoprenoids
and also production of non-volatile defense compounds such as polyphenols. b. The octadecanoid signaling pathway for some gene expression in tea leaves: Exogenous
MeJA could in a great degree lead to the activation of lipoxygenase pathway that results
in release of green leaf volatiles (a variety of C
₆
aldehydes) and synthesis of jasmonate and methyl jasmonate which could further elicit
the JA pathway in the whole tea plant

According to the results of RNA-Seq, the expressions of 10 DEGs related to the terpenoids
backbone biosynthesis pathway were upregulated by treatment of MeJA (Table 3). The content isopentenyl diphosphate should be promoted by the higher expression
level of Hydroxymethylglutaryl-CoA reductase (HMG-CoA) (EC:1.1.1.34) mRNA, which was
increased by 2.88-fold after MeJA_24h treatment. The increased expression of ispH
(EC:1.17.1.2) mRNA could increase the biosynthesis of IDP and DMADP. The high expression
of GGPS (EC:2.5.1.1 2.5.1.10 2.5.1.29) mRNA, which was increased by 4.79-fold after
MeJA treatment, could also promote the synthesis of GDP, GGDP, and FDP (Additional
file 6: Figure S4, Additional file 7: Figure S6).

According to our metabolite results, we found that the levels of the above-mentioned
flavor aroma compounds were higher in treated compared with untreated samples (Table 1). In particular, Linalool and Geraniol, which contribute significantly to tea aroma
quality with a floral smell, increased by 1.91 and 2.63 ?g/2 g, respectively.

The accumulation of GDP, GGDP, and FDP, could promote the production of terpenoids
biosynthesis (C
₁₀
–C
₄₀
). Note that the expression level of terpene synthase (TPS), which is an important
hydrolyzing enzyme for releasing tea aroma, showed no significant difference in expression
between MeJA-treated tea leaves the controls. Linalool and Geraniol are synthesized
from the precursors GDP, GGDP, and FDP; therefore, speculated that the contents of
these precursors were the limiting factors for aroma volatiles release from tea leaves.
The MeJA treatment significantly increased terpenoids biosynthesis by upregulating
the expressions of genes related to the terpenoids backbone biosynthesis pathway.

MeJA affects phenylpropanoids and some amino acid-derived volatiles biosynthesis pathways

Aldehydes and alcohols derived from the degradation of branched-chain and aromatic
amino acids constitute a class of highly abundant volatiles in tea; however, their
metabolic pathways have been barely analyzed. The catabolism of amino acids has been
analyzed in detail, and is initiated by amino transferases forming 2-ketoacids that
serve as substrates for three biochemical reactions: (i) oxidative decarboxylation
to carboxylic acids; (ii) decarboxylation to aldehydes; and (iii) reduction to 2-hydroxyacids.
Compounds derived from phenylalanine, such as phenylacetaldehyde and 2-phenylethanol,
are abundant in various fruits, such as strawberry, tomato and grape, and in tea 17], 56].

Phenylpropanoids/benzenoids and volatile compounds, primarily derived from phenylalanine,
contribute to the aromas and scents of many plant species and play important roles
in plant communication with the environment 57], 58]. Treatment by MeJA affected the phenylpropanoids biosynthesis pathway. The expression
of phenylalanine ammonia-lyase (EC:4.3.1.24) increased by 2.14-fold, which could lead
directly to the production of more Cinnamic acid; the high content of this precursor
ensures sufficient substrates to produce benzaldehyde and benzylalcohol. The high
expression level of beta-glucosidase in this pathway could lead to a greatly increased
content of coumarin (Table 3; Additional file 5: Figure S3, Additional file 8: Figure S5). Moreover, phenylethyl alcohol and methyl salicylate are common components
of floral scents in plants 59]. During the first 12 h, these two compounds were massively synthesized, which would
affect the quality of tea aroma.

MeJA affects Carotenoid-derived volatiles biosynthesis pathways

Carotenoid-derived volatiles also contribute to the aroma and quality of tea. The
transcriptome results showed that at least seven DEGs involved in the carotenoid pathway
were affected by MeJA treatment. The expressions of crtB, PDS and NCED increased by
2.76-, 3.45- and 6.16-fold, respectively, in 24 h MeJA-treated tea leaves compared
with the controls. Increased expression of these three DEGs would result in upregulated
biosynthesis of ?-carotene (Tables 1 and 3).

Validation of some important DEGs profiling using RT-qPCR

In order to experimentally validate the reliability of these important differential
expressed genes obtained from the assembled transcriptome and profiling of gene expression
obtained by RNA-Seq data, a total of 11 key unigenes involved in the biosynthesis
of ?-linolenic acid degradation (LOX2S, AOC, JOM, acyl-CoA oxidase) and terpenoid
backbones biosynthesis (chlP, GGPS, DHDDS, DXS, 4-hydroxy-3-methylbut-2-enyl diphosphate
reductase) and some other important pathways (all-trans-nonaprenyl-diphosphate synthase,
trans-cinnamate 4-monooxygenase, and branched-chain amino acid aminotransferase) were
selected for RT-qPCRs (Fig. 5).

Fig. 5. Quantitative RT-qPCR validations. A total of 11 genes were selected for the quantitative
RT-qPCR experiments. Of them, AOC(allene oxide cyclase), chlP(geranylgeranyl reductase),
JOM(jasmonate O-methyltransferase), LOX2S(lipoxygenase), GGPS(geranylgeranyl diphosphate
synthase, type II), DHDDS(ditrans,polycis-polyprenyl diphosphate synthase) and DXS(1-deoxy-D-xylulose-5-phosphate
synthase), acyl-CoA oxidase, all-trans-nonaprenyl-diphosphate synthase, 4-hydroxy-3-methylbut-2-enyl
diphosphate reductase, trans-cinnamate 4-monooxygenase, and branched-chain amino acid
aminotransferase

The results suggest that the assembled transcripts are reliable and the designed primer
pairs are suitable for the subsequent expression experiments. Based on the delta-delta
Ct (2-??Ct) method, relative expression levels of the selected unigenes were calculated
and compared among the four different tissues. Mostly, the expression patterns of
these genes detected by RT-qPCR were mainly consistent with those from RNA-Seq data.
Overall, RT-qPCR experiments confirmed that the unigenes obtained from the assembled
transcriptome are trustworthy and gene expression profiles from RNA-Seq data should
be believable.