Abstract
Trichothecium roseum is a harmful postharvest fungus causing serious
damage, together with the secretion of insidious mycotoxins, on apples,
melons, and other important fruits. Cuminal, a predominant component of
Cuminum cyminum essential oil has proven to successfully inhibit the
growth of T. roseum in vitro and in vivo. Electron microscopic
observations revealed cuminal exposure impaired the fungal morphology
and ultrastructure, particularly the plasmalemma. Transcriptome and
proteome analysis was used to investigate the responses of T. roseum to
exposure of cuminal. In total, 2825 differentially expressed
transcripts (1516 up and 1309 down) and 225 differentially expressed
proteins (90 up and 135 down) were determined. Overall, notable parts
of these differentially expressed genes functionally belong to
subcellular localities of the membrane system and cytosol, along with
ribosomes, mitochondria and peroxisomes. According to the localization
analysis and the biological annotation of these genes, carbohydrate and
lipids metabolism, redox homeostasis, and asexual reproduction were
among the most enriched gene ontology (GO) terms. Biological pathway
enrichment analysis showed that lipids and amino acid degradation,
ATP-binding cassette transporters, membrane reconstitution, mRNA
surveillance pathway and peroxisome were elevated, whereas secondary
metabolite biosynthesis, cell cycle, and glycolysis/gluconeogenesis
were down regulated. Further integrated omics analysis showed that
cuminal exposure first impaired the polarity of the cytoplasmic
membrane and then triggered the reconstitution and dysfunction of
fungal plasmalemma, resulting in handicapped nutrient procurement of
the cells. Consequently, fungal cells showed starvation stress with
limited carbohydrate metabolism, resulting a metabolic shift to
catabolism of the cell’s own components in response to the stress.
Additionally, these predicaments brought about oxidative stress, which,
in collaboration with the starvation, damaged certain critical
organelles such as mitochondria. Such degeneration, accompanied by
energy deficiency, suppressed the biosynthesis of essential proteins
and inhibited fungal growth.
Keywords: transcriptome, proteome, Trichothecium roseum, antifungals,
cuminal, postharvest
1. Introduction
Postharvest fungal diseases are responsible for quality deterioration
of fruits and vegetables and for resultant severe losses of produce
during handling, transportation, and storage. Phytopathogen
Trichothecium roseum is one of the most significant spoilage fungi in
China, causing decay in stored apples [[34]1], melons [[35]2,[36]3],
grapes [[37]4], strawberries [[38]5], and oranges [[39]6]. Presently
the pathogen can be controlled predominantly by chemical fungicides
[[40]7]. Nonetheless, decline in the number of available fungicides to
be used for postharvest treatment, reduced efficacy due to the
emergence of pathogen resistance to these chemicals, and the growing
public concerns over chemical residues in food and the environment have
necessitated the seeking of alternative solutions to control the fungal
spoilage and to guarantee food safety and consumer health [[41]8].
Essential oils (EOs), generally recognized as safe by the US Food and
Drug Administration [[42]9], are natural substances with established
antimicrobial activities [[43]10,[44]11] and are a candidate instrument
for controlling postharvest disease [[45]12,[46]13]. Nonetheless, this
candidacy is compromised by the expensiveness of these natural
substances due to their generally low yields. However, exploring their
mode of actions is valuable to the development of novel alternatives.
The multicomponent EOs attribute their mode of action to various
responsible components [[47]14,[48]15] rather than a simple and
sole-mechanism action of an individual component. This wide mode action
is an advantage of EOs over synthetic fungicides, but at the same time,
it complicates the elucidation of the antifungal mechanisms of EOs. For
this reason, the search for the antifungal mechanism of the active
component of the EOs is of practical significance. Such pioneer
research work on eugenol and cinnamaldehyde revealed their effect on
critical enzymes, such as those involving fungal bioenergetics
[[49]16,[50]17]. Nonetheless, the earlier biological events elicited by
these EO components were not well understood. However, such an
understanding might be of primary importance for the elucidation of the
action modes of these compounds.
Conventionally, the complex antifungal mechanisms of EOs were basically
attributed to their lipophilicity. When interacting with cells, EOs can
damage the cytoplasmic membrane by causing leakage of intracellular
substances, thereby resulting in cell death [[51]18]. Reports have
indicated an effect of EOs on a decrease of energy acquisition
following the sabotage of fungal mitochondria by enhanced accumulation
of reactive oxygen species (ROS) [[52]19,[53]20]. Indeed, these
findings are in line with some action modes of commercial antibiotics
[[54]21]. Accordingly, exploring novel targets could be an instrument
for developing safer fungicides with fewer and mitigated side effects.
The investigation of the interaction of fungal cells with antifungal
EOs agents may provide a global analysis of cellular gene expression
alteration upon treatment. Such analysis could give us opportunities to
fully understand fungal responses to these compounds. Transcriptome
sequencing and iTRAQ (isobaric tags for relative and absolute
quantitation) proteomic sequencing can explore gene expression changes
at transcript and translational levels, respectively, occasioned by
different stimuli [[55]22,[56]23,[57]24]. Given the post-transcript
regulation, protein turnovers, and alternative translation rate,
consolidation of transcriptome and proteome analyses, along with
bioinformatics interpretation, could furnish us with a more thorough
understanding of the gene networks involved [[58]25] and of molecular
events during the interaction of fungi with antifungals.
Cuminum cyminum EO and its predominant component cuminal
(4-Isopropylbenzaldehyde) showed antifungal activity against T. roseum
[[59]26], providing us with a potential way to control fruit rot caused
by this devastating pathogen. Nonetheless, their antifungal mechanisms
have not yet been well investigated. In the present investigation, the
antifungal activity of cuminal against T. roseum was assessed. RNA-seq
transcriptome and iTRAQ proteome analysis were conducted on T. roseum
upon cuminal exposure to elucidate the wide metabolic responses of the
fungus to cuminal. Analysis of the fungal responses indicated that the
treatment impaired the polarity of cytoplasmic membrane and triggered
the reconstitution and dysfunction of the membrane, resulting in
handicapped nutrients procurement and fungal starvation, therefore
causing the inhibition of fungal growth.
2. Materials and Methods
2.1. In Vitro Antifungal Activity Measurement
2.1.1. Antigermination Test and Minimum Inhibitory Concentration (MIC)
The modified method of Chitarra et al. [[60]27] was used to evaluate
the effect of cuminal on T. roseum conidia germination. T. roseum
(Pers.: Fr.) Link was isolated from decayed muskmelon fruit with
typical pink rot and preserved on potato dextrose agar (PDA) at 25 °C.
The fungal conidia were washed from the surface of PDA plates with
sterile 0.85% saline containing 0.1% Tween 80 (v/v) to prepare conidia
inoculum and stored at 4 °C for further use. Cuminal (Sigma-Aldrich,
Shanghai) was added to potato dextrose broth (PDB) by dissolving the
requisite amounts of cuminal into PDB containing 0.05% (v/v) Tween 80
to prepare a series of concentrations (0, 0.05, 0.10, 0.15, 0.20, and
0.25 µL/mL), and a 0-concentrate one was used as the control. Slides
inoculated with 20 µL conidia in the above PDB medium (10^6 conidia/mL)
were placed on moist filter paper in Petri plates, and the plates were
parafilmed to avoid evaporation and incubated at 27 °C. When the
germination rate of the control conidia reached 80, all of the
corresponding treatment conidia on the slides were immediately fixed
with lactophenol cotton blue to stop further germination and the
germinated conidia of each slide were counted. The results were
expressed as the antigermination rate using the formula:
[MATH:
GI(%)=
[(Gc−Gt)/Gc]×100 :MATH]
(1)
where G[c] and G[t] represent the mean number of germinated conidia in
the control and treated slides, respectively. Each treatment was
performed in triplicate.
Minimum inhibitory concentration (MIC) of cuminal against T. roseum in
PDB was determined by a serial dilution techniques [[61]28] using
96-well microtitre plates. Cuminal was added in PDB with inoculum and
the microplates were incubated at 27 °C for 72 h. The lowest
concentration without visible growth under binocular microscope was
defined as MIC.
2.1.2. Antifungal Activity of Cuminal Vapor against Mycelial Growth
In vitro antifungal assays of cuminal vapor were carried out using a
modified method of Aguilar-Gonzaliez et al. [[62]29]. Briefly, Mycelial
disks (4-mm-diameter) from the periphery of 7-day-old cultures were
centrally inoculated onto the PDA plates with the mycelium surface
facing down. A piece of round Whatman No.1 filter paper
(20-mm-diameter) was stuck to the inside center of each Petri dish lid
and requisite amounts of cuminal were poured on each round paper to
achieve concentrations of 0, 0.0625, 0.125, 0.25, 0.50, and 1.00 µL/mL
relative to the air volume in the Petri dishes, and then plates were
parafilmed to avoid vapor loss and incubated at 27 °C in the dark for 7
days. Among the plates, the 0-concentrate ones were used as control.
The mean of two perpendicular diameters of the colony was measured. The
rate of mycelial radial growth inhibition was calculated with formula:
[MATH:
MGI(%)
=[(C−T)/C]×100 :MATH]
(2)
where C and T represent mycelial growth diameter in control and
cuminal-added Petri plates, respectively. Three plates were used for
each treatment as replications.
2.2. In Vivo Antifungal Activity
The in vivo antifungal activity of cuminal against T. roseum was tested
on apple fruits using a modified method from the literature [[63]28].
Apple fruits (cv. Fuji) were obtained from an orchard located in
Jinning town, Gansu (China). Fruits free of wounds and rot, and as
homogeneous in maturity and size as possible were selected. Fruits were
surface-disinfected by immersion in a 2% sodium hypochlorite solution
for 6 min, washed twice by immersion in sterile deionized water, and
air-dried in shade to remove excess surface water. The fruits were
wounded (3 mm deep and 3 mm wide) with a sterile nail at the equator,
with two directly opposite wounds per fruit. Each treatment had two
replicates with eight fruits per replicate. Cuminal was added to PDB
containing 0.05% Tween 80 to reach cuminal concentrations of 0, 0.5MIC
(2 μL/mL), and MIC (4 μL/mL), and T. roseum conidia were added to the
above PDBs to reach a concentration of 10^6 conidia/mL. The sets of
conidia in PDBs were cultured in duplicate in a shaker (130 r/min) at
27 °C for 3 h. To investigate the sustaining efficacy of the cuminal
treatment on T. roseum conidia, one copy of the above conidia set was
centrifuged to remove cuminal-containing PDB and the harvested conidia
were washed twice with distilled and sterile water by re-suspension and
centrifugation. The washed conidia were re-suspended in fresh PDB to
reach concentration of 10^6 conidia/mL. Then, 20 μL of unwashed- and
washed-conidia PDBs was placed into each wound, separately. The apple
fruits were put into plastic boxes with sterile water to maintain a
high relative humidity (95%) and kept at 25 °C for observations. The
mean of perpendicular lesion diameters of each wound was measured on
day 14.
2.3. Electron Microscope Observations
One-hour-old PDB culture of T. roseum conidia was exposed to 4 µL/mL of
cuminal while the control was without cuminal, and further cultured for
1 day. The fungal cells were harvested, and washed thrice with sterile
0.85% saline. The morphological and ultrastructural changes were
observed under scanning electron microscopy (SEM) (Evo-18, Zeiss) and
transmission electron microscopy (TEM) (Tecnai G2-T20 STwin 200kV, Fei
Co.), respectively, after sample preparation was done according to the
method of Dwivedy et al. [[64]30]. Digital images were acquired with a
charge-coupled device camera (Megaview III, Fei Company, Eindhoven, The
Netherlands) using the microscope-attached software iTEM (Sift Imaging
System, Münster, Germany).
2.4. Fungal Sample Preparation
The T. roseum conidia were incubated in 1000 mL Erlemeyer flasks
containing 200 mL PDB inoculated with 100 μL conidia suspension (5 ×
10^5 conidia/mL), at 25 °C with shaking (200 r/min) for 96 h. Then, the
flasks were supplied with fresh sterile PDB (200 mL) with 0.1% (v/v)
Tween 80. Cuminal was consecutively added to certain flasks to reach a
final concentration of 4 μL/mL (v/v). Flasks without cuminal addition
were used as control. All flasks were cultured for a further 2 h under
the same conditions as before. The mycelia were harvested by vacuum
filtration on a sterile clean bench, washed thrice with sterile
deionized water, and snap-frozen with liquid nitrogen. Three replicate
cultures were performed for each treatment and control. All samples
were stored at −80 °C until omics analysis.
2.5. RNA Sequencing and Data Processing
2.5.1. RNA Extraction and Sequencing Strategies
Total RNA was extracted with TRIzol reagent (Life Technologies, UK)
from the prepared T. roseum mycelia samples. The libraries were
sequenced on the Illumina HiSeq™2500 platform. Readings were analyzed
by tool FASTQC, and the data of poor quality bases (phred ≥20),
unexpected Illumina adapters and poly-A tails were removed using the
Toolkit NGS QC v2.3.3 [[65]31].
2.5.2. Transcriptome Assembly and Annotations
De novo short read assembly was carried out with the software of Tophat
and Cufflinks. The assembled readings were mapped to the complete
genome of Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
(Baker’s yeast) using Tophat and Bowtie2. Unigenes were aligned to the
NCBI NR Database, wherein the unigenes encoding proteins with high
similarity (e < 1 × 10^−5) to the known proteins were used for
annotation. Gene ontology (GO) annotation was conducted with Blast2GO
software. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation was
performed with the database [66]http://www.genome.jp/kegg/ pathway.html
[[67]32].
2.5.3. Transcriptome Analysis
As described by Yan et al. [[68]32], differential transcript
accumulation in samples of controls and treatments was observed using
bowtie2 and eXpress. The gene expression levels were calculated with
fragments per kilobase per million method. Differentially expressed
genes (DEGs) were judged based on Baggerley’s test with a significance
level of <0.05 and the fold change >2 or <0.5. GO enrichment analysis
of DEGs was performed by mapping them to GO terms in the database
(Available online: [69]http://www.geneontology.org/) and by calculating
gene numbers for every term, and by hypergeometric testing to determine
significantly enriched GO terms. KEGG pathway enrichment analysis was
conducted on DEGs.
2.6. iTRAQ-Labeled Proteome Analysis
2.6.1. Protein Extraction, Digestion and Labeling
Total mycelia proteins were extracted as described by Zhang et al.
[[70]23]. Briefly, the fungal samples were re-suspended in lysis buffer
supplemented with protease inhibitor solution, and the suspensions were
sonicated on ice for 15 min. After centrifugation at 25,000× g for 20
min at 4 °C, the sediments were discarded, and the expected proteins in
supernatants were quantified and stored at −70 °C until digestion.
As described by Wisniewski et al. [[71]33], proteins were digested
using the filter-aided sample preparation method. iTRAQ labeling was
performed using iTRAQ 8-plex reagent kits (AB Sciex, Framingham, MA,
USA). iTRAQ reagent was rested at room temperature and centrifuged to
the tube bottom, and isopropanol was added. iTRAQ reagent (100 μL) was
transferred to the sample tubes and vortexed before spinning. The tubes
were incubated at room temperature for 2 h and 200 µL of water was
added to quench the labeling. The solutions were lyophilized and stored
at −70 °C for analysis.
2.6.2. Peptide Fractionation and Mass-Spectrometry Analysis
Peptide fractionation and mass-spectrometry analysis were routinely
carried out as described by Zhang et al. [[72]34], and the
enzymatically hydrolyzed protein solution was re-suspended with 110 µL
of eluent A (acetonitrile-H[2]O-formic acid, 2:98:0.1, v/v/v). Peptides
were separated using an Agilent 1200 HPLC (Wilmington, DE, USA) with a
guard column and a separation column, and UV detection wavelengths were
210 nm and 280 nm. Mass-spectrometry analysis was performed on a Triple
TOF 5600 System (AB SCIEX, Foster City, CA, USA) fitted with a
Nanospray III source (AB SCIEX, Framingham, US). Data was acquired
under the following conditions: ion spray voltage 2.4 kV, 35 PSI
curtain gas, 5 PSI nebulizer gas, and interface heater temperature 150
°C. The MS scans were acquired in 250ms, and each cycle time was fixed
to 2.5s. The dynamic exclusion set was 22s.
2.6.3. Protein Identification and Quantification
The MS/MS spectra were processed using Protein Pilot Software v.5.0 (AB
Sciex, Framingham, MA, USA) as described by Zhang et al. [[73]34].
Laconically, only peptides with the 95% confidence interval were
counted as the identified protein. Proteins with an average fold change
>1.2 or <0.83 in treated groups compared to controls were regarded as
differentially expressed proteins (DEPs). The experimental data from
MS/MS were matched with the theory data to achieve protein
identification with the following parameters: sample type, iTRAQ 8plex;
cys alkylation, iodoacetamide; instrument, Orbi MS (sub-ppm) and Orbi
MS/MS; and the search effort was thorough.
2.6.4. Bioinformatics Analysis of DEPs
Homology mapping of the identified proteins to S. cerevisiae (ATCC
204508/S288c) was conducted based on sequence similarity, and their
biological function information analysis was carried out through
comparative analysis. An integrated GO enrichment and KEGG pathway
analysis of DEPs was performed with OmicsBean (Available online:
[74]http://www.omicsbean.cn). The GO term or pathway enrichment of DEPs
were considered as significant when p < 0.05 [[75]35]. To examine
possible post-transcriptional regulation and to achieve in-depth
understanding of the proteome and transcriptome data, correlation
analysis was conducted.
2.7. Protein–Protein Interaction Analysis
For DEPs and the proteins encoded by DEGs involved in the specific KEEG
pathways, their predicted protein–protein interactions (PPI) were
analyzed from the STRING database [[76]36]. All these interactions were
visualized using the Cytoscape tool.
2.8. Statistical Analysis
All antifungal tests were conducted in triplicate; omics analysis was
with 3 biological replicates. Results were statistically processed and
subjected to analysis of variance. Means significantly different were
separated by the Tukey test using SPSS version 2019.
3. Results
3.1. Antifungal Activity in Vitro
In PDB medium containing various concentrations of cuminal (0.05 to
0.25 µL/mL), the inhibition of the conidial germination was observed
when the effect was in a dose dependent manner ([77]Figure 1A). In the
bioassay matrix, the cuminal obtained an inhibition rate of 30.2% at
the lowest concentration 0.05 µL/mL. When the concentration elevated,
the antigermination efficacy was increased. However, at relatively
higher concentrations, the ascending of the concentration showed no
significant boost of antigermination rate. A maximum rate of 96.1% was
observed at 0.25 µL/mL of cuminal.
Figure 1.
[78]Figure 1
[79]Open in a new tab
In vitro antifungal activities of cuminal against T. roseum. (A)
Antigermination effect against T. roseum conidia. (B) Vapor contact
inhibition of mycelial growth. Bars with different letters indicate
mean values significantly different at p < 0.05 according to Tukey
test. Data are expressed as mean of three replicates ± SD.
Via a modified microdilution technique, the MIC of cuminal against T.
roseum in PDB medium was established to be 4 µL/mL. This data was used
in the implementation of in vivo test and fungal sample preparation for
the following omics analysis.
By virtue of the practical value of the essential oil fumigation, the
inhibition effect of cuminal vapor against T. roseum mycelia was
tested. Considering the effective vapor concentrations were not in
proportion to the added cuminal doses, an equal-ratio gradient
concentration set was used in the measurement of the vapor contact
inhibition of mycelial growth. Under this bioassay matrix, the
elevation of cuminal concentrations obtained stable increase of the
mycelial growth inhibition ([80]Figure 1B), where the lowest
concentration (0.0625 µL/mL) showed an inhibition of 12.6%, and the
concentration of 0.5 µL/mL reached an inhibition of 99.3%, with no
significant increase when the concentration was further doubled.
3.2. In Vivo Apple Fruit Assay
The results on the artificially inoculated apple fruits with
cuminal-treated T. roseum cells showed a noticeable suppression of the
fungal growth by this agent in the host matrix ([81]Figure 2). The
lesion diameters in the treated apple fruits were significantly shorter
(p < 0.05) than that of the control sets. The in vivo assay showed that
both cuminal treatments at 0.5MIC and MIC well demonstrated an
antifungal effect on apple fruits. Notably, the in vivo assay disclosed
the sustained inhibition of the growth of T. roseum by cuminal
treatment: after removing the surplus cuminal by washing with the
saline, the treatment cells were still devitalized on the apple fruits.
This was an interesting finding; the in vivo assay, unlike most other
similar in vivo assays, indicated that cuminal treatment might disorder
the fungal cells and that removing the remaining cuminal in the matrix
cannot revive their original vitality.
Figure 2.
[82]Figure 2
[83]Open in a new tab
In vivo antifungal effect of cuminal against T. roseum on apple fruits.
Bars with different letters indicate mean values significantly
different at P < 0.05 according to Tukey test. Data are expressed as
mean of three replicates ± SD.
3.3. Effects on Conidial Morphology and Ultrastructure
In SEM analysis, the conidia of T. roseum exposed to cuminal were found
inflated and distorted in comparison to the control set ([84]Figure
3A,B). Obvious roughness was seen on the conidial surface, indicating
that the relationship between cell wall and plasmalemma was changed
upon cuminal exposure, which denotes the membrane as the site of
action. Upon exposure to cuminal, conspicuous deformities of cellular
inner structures and organization were observed in TEM ([85]Figure
3C,D). In treatment cells, detachment of the plasmalemma from the cell
wall occurred with formation of small lomasomes and decrease in the
cell matrix, possibly due to the decreased thickness of plasmalemma
tentatively caused by the reconstitution of the plasmalemma. Another
contingent event of the decreasing of plasmalemma thickness was the
reduction of the cell matrix on account of the leakage of cellular
components. Moreover, manifest decomposition of the cellular organelles
and decline of the inner organization were observed in line with the
debilitated cell vigor, shown both in in vitro and in vivo tests.
Notably, abnormal cytoplasm coagulation indicated by high electron
density was imaged, suggesting an unconquerable oxidative stress
suffered in the treatment cells.
Figure 3.
[86]Figure 3
[87]Open in a new tab
Morphological (A: control, and B: treatment under scanning electron
microscopy) and ultrastructural (C: control, and D: treatment under
transmission electron microscopy) changes of T. roseum conidia upon
cuminal exposure. (CW: cell wall, FL: fibril layer, PM: plasmalemma, M:
mitochondrion, S: septum, V: vacuole, N: nucleus, L: lomasome).
3.4. Fungal Transcriptome Profiles upon Cuminal Exposure
In total, 14897 unigenes were assembled after de novo transcript
splicing ([88]Table S1), and among them, 2825 genes were differentially
expressed genes. Cuminal exposure up regulated 1516 genes and down
regulated 1309 genes ([89]Figure 4A,B). For these DEGs, 248 genes were
highly up regulated (>10-fold change) at the transcriptional level,
including 112 genes that were almost not transcripted in the control
samples (the fold change values were marked as ‘inf’ in the
[90]supplementary material). Meanwhile, 381 genes were deeply down
regulated with fold-changes <0.1, including 78 genes barely even
expressed in the cuminal treated samples at transcriptional level
([91]Table S2).
Figure 4.
[92]Figure 4
[93]Open in a new tab
Cuminal exposure regulated gene expression of T. roseum at mRNA level.
(A) The volcano plot of detected genes indicating significantly up
(red) and down (green) regulated genes. (B) Comparison of the numbers
of all identified genes and differentially expressed genes (up vs. down
regulated).
3.5. GO Analysis of DEGs
The DEGs were divided into up and down subgroups. Most of the DEGs were
annotated with GO terms according to their functions, and these terms
were mapped into “biological processes”, “cellular components”, and
“molecular functions” categories. For up regulated DEGs, the terms most
associated were metabolic process, cellular process, cell, cell part,
and catalytic activity. Most enriched GO terms of up DEGs were
single-organism process (315, 20.78%), single-organism cellular process
(290, 19.13%), single-organism metabolic process (219, 14.45%), and
catabolic process (102, 6.73%) in the biological process category.
Additionally, genes involved in oxacid metabolism (70, 4.62%) and
oxi-reduction process (67, 4.42%) were also up regulated. High
enrichment in the subcellular components localized in the cell (342,
22.56%), cell part (341, 22.49%), intracellular (320, 21.11%), and
intracellular part (315, 2.078%). Also, the mitochondrion (73, 4.82%),
mitochondrial part (54, 3.56%), and mitochondrial matrix (26, 1.72%)
were enriched with a noticeable amount of up regulated DEGs. In the
molecular function category, catalytic activity (228, 15.04) and ion
binding (166, 10.95%) were the most modulated entries involving the up
regulated DEGs. Besides ion binding, a large part of the up regulated
DEGs were also involved in the binding related function of other
molecular groups including cofactors and other small molecules
([94]Figure S1).
The situation of GO analysis of down DEGs was different from that of up
DEGs. Although most of the down DEGs were still involved in the
biological process of metabolic process (262, 20.02%) and
single-organism cellular process (236, 18.03%), several key processes
including oxidation-reduction process (50, 3.82%), negative regulation
of cell cycle (20, 1.53%), post replication repair (8, 0.6%), negative
regulation of cell cycle process (15, 1.15%), and metabolic process
relating to nucleotide and nucleoside derivatives were also enriched
with a noticeable quantity of down regulated DEGs, and these processes
are crucial to fungal growth. Most of the down regulated DEGs belong to
cellular components of cell part (277, 21.16%), cell (277, 21.16%),
intracellular part (258, 19.71%), intracellular (259, 19.77%), and
cytoplasm (212, 16.20%), but replication fork, protein–DNA complex,
ribo–nucleoprotein granule, septin cytoskeleton, and mini-chromosome
maintenance protein complex were the related cellular components of a
noticeable number of down regulated DEGs too. As to the molecular
functions of the down DEGs, most of them were mapped into catalytic
function and binding with other molecules, including entries of
nucleotide and nucleoside derivatives binding ([95]Figure S2).
3.6. KEGG Metabolic Pathways of DEGs
To investigate the enrichment situation of the DEGs in metabolic
pathways, these genes were mapped into the KEGG pathways with the
Blast2GO program. In total, the 2825 DEGs were mapped into 216
metabolic pathways in which biosynthesis of amino acids (202, 7.15%),
carbon metabolism (180, 6.37%), and ribosome (164, 5.81%) were the top
three ones. For clearer understanding the effects of cuminal exposure
on the fungal metabolism, the KEGG enrichment of DEGs was analyzed for
up and down regulated DEGs, separately ([96]Figure 5). A noticeable
number of up regulated genes enriched the metabolic pathways (89%),
biosynthesis of secondary metabolites (45%), biosynthesis of
antibiotics (41%), carbon metabolism (19%), and biosynthesis of amino
acids (18%). In particular, citrate cycle, pyruvate metabolism,
glycerophospholipid metabolism, mRNA surveillance pathway and
peroxisome were also enriched with notable DEGs. From an upper
hierarchical view, the top enriched KEGG ways were global and overview
maps (A0), carbohydrate metabolism (AA) and other unknowns (HA)
([97]Figure 5A). For down regulated DEGs, the top enriched KEEG ways
were metabolic pathways (84%), biosynthesis of secondary metabolites
(37%), antibiotics biosynthesis (34%), cell cycle (17%), and
glycolysis/gluconeogenesis (12%). From an upper hierarchy, most of
these down regulated DEGs function in global and overview maps (A0),
carbohydrate metabolism (AA), cell growth and death (DC), and other
unknowns (HA) ([98]Figure 5B).
Figure 5.
[99]Figure 5
[100]Open in a new tab
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of
differentially expressed genes (DEGs) and hierarchical belonging of
each enriched pathway to upper level pathways. (A) Pathway
classification of up regulated DEGs. (B) Pathway classification of down
regulated DEGs.
To evaluate the effects of cuminal exposure on the different KEGG
pathways of T. roseum, functional enrichment analyses, where an index
(rich factor) can evaluate how severely the treatment interferes in the
pathways, were conducted ([101]Figure 6). The rich factor indicates the
ratio of the number of DEGs in a pathway to the number of genes
annotated in the pathway. Higher rich factors define greater degrees of
enrichment. ABC (ATP-binding cassette) transporters, lipid metabolism,
valine, leucine and isoleucine metabolism, steroid biosynthesis,
tryptophan metabolism, and propanoate metabolism had greater enrichment
of up regulated DEGs. Additionally, certain large pathways such as
metabolic pathways embraced a larger number of up regulated DEGs, but
their enrichment factors were not high. Certain key pathways such as
citrate cycle, pyruvate metabolism, and glycerophospholipid metabolism
have both considerable rich factors and DEG numbers ([102]Figure 6A).
Figure 6.
[103]Figure 6
[104]Open in a new tab
Rich-factor scatter plot of select pathway enriched with DEGs. (A) Up
regulated DEGs. (B) Down regulated DEGs. Rich factor is the number
ratio of differentially expressed genes to total annotated genes in a
pathway. Dot areas show the number of DEGs, and the selection was based
on the KEGG enrichment situation and pertinence to the metabolic blocks
underpinning the fungal response to cuminal exposure.
The situation of functional enrichment of down regulated DEGs was
different from that of up regulated DEGs. Concisely, the advanced
glycation end-product and receptor for advanced glycation end-product
signaling pathway and hippo signaling pathway were with higher in rich
factors. Tyrosine metabolism, starch and sucrose metabolism, fatty acid
degradation, DNA replication, and glycolysis/gluconeogenesis were the
second enriched group of pathways, both with considerable rich factors
and DEG numbers ([105]Figure 6B).
3.7. Global Proteome Changes in Response to Cuminal Exposure
Using the Mascot search engine, the iTRAQ LC-MS/MS investigation
generated 94463 spectra and identified 1485 proteins, in which 225 were
differentially expressed ([106]Table S3). Among these DEPs, 90 were up
regulated while 135 were down regulated ([107]Figure 7).
Figure 7.
[108]Figure 7
[109]Open in a new tab
Cuminal exposure regulated gene expression of T. roseum at protein
level. (A) The volcano plot of detected proteins indicating
significantly up (red) and down (green) regulated proteins. (B)
Comparison of the numbers of all identified proteins and differentially
expressed proteins (up vs. down regulated).
As regards the magnitude of regulation, 21 proteins were highly up
regulated with a fold-change >2, and 11 proteins were extremely low
expressed with a fold-change <0.5. Of these DEPs, phosphatidylserine
decarboxylase proenzyme 1 (mitochondrial) (PSD1) was the most highly
expressed, with an increase of more than 17-fold and aspartate-tRNA
ligase (cytoplasmic) (DPS1) increased by nearly 10-fold. In contrast,
checkpoint serine/threonine-protein kinase (BUB1), an enzyme involved
in cell cycle checkpoint enforcement, was the lowest expressed protein,
decreasing by nearly 7-fold.
3.8. GO Annotation Analysis of the DEPs
Of the 225 DEPs, 212 were annotated in GO terms classified into
biological process, cellular components and molecular function. The
prominently enriched GO terms are demonstrated in [110]Figures S3 and
S4. Most of the 90 up regulated DEPs were mainly involved in small
molecular metabolic process (15, 16.67%), carboxylic acid metabolic
process (10, 11.11%), oxoacid metabolic process (10, 11.11%), organic
acid metabolic process (10, 11.11%), small molecule biosynthetic
process (7, 7.78%) and cellular amino acid metabolic process (6,
6.67%). Certain other up regulated DEPs enriched the metabolism of
carbohydrate and amino acid derivatives. In terms of cellular
components, the up regulated DEPs were mainly localized in cell part
(30, 33.33%), cytoplasm (26, 28.89%), mitochondrial matrix (4, 4.44%),
mitochondrial protein matrix (3, 3.33%), mitochondrial nucleoid (2,
2.22%) and some mitochondrial complexes. Regarding the molecular
function enrichment of these up regulated DEPs, the most predominant
enrichment was catalytic activity (27, 30%), followed by carboxylic
ester hydrolase activity (3, 3.33%), transferring nitrogenous activity
(2, 2.22%), and transaminase activity (2, 2.22%). Other molecular
functions of up regulated DEPs were diverse and beyond the list
([111]Figure S3). For biological processes, the 135 down regulated DEPs
mainly enriched the organonitrogen compound metabolic process (26,
19.26%), cellular biosynthetic process (25, 18.52%), organic substance
biosynthetic process (25, 18.52%) and organonitrogen compound
biosynthetic process (20, 14.82%). Other biological processes enriched
with the down regulated DEPs included translation, ribosome biogenesis
and rRNA processing. These down regulated DEPs noticeably localized in
the cellular components of intracellular (32, 23.70%), intracellular
part (32, 23.70%), cytoplasm (29, 21.48%), cytoplasm part (25, 18.52%),
intracellular organelle part (24, 17.78%), macromolecular complex (22,
16.30%), intracellular non-membrane-bounded organelle (20, 14.81%) and
non-membrane-bounded organelle (20, 14.81%). Additionally, ribosome
related components were also well enriched with down regulated DEPs.
The molecular functions of these down regulated DEPs included the
structural constituent of ribosome (15, 11.11%), structural molecule
activity (15, 11.11%), RNA binding (10, 7.41%) and others such as
transferase activity ([112]Figure S4).
3.9. KEGG Pathway Analysis of DEPs
The Blast2GO KEGG enrichment analysis showed that the most
significantly enriched pathways of some of the 90 up regulated DEPs
were metabolic pathways (13%), biosynthesis of secondary metabolites
(10%) and biosynthesis of antibiotics (8%). Particularly, carbon
metabolism, citrate cycle and fatty acid degradation were well enriched
with certain up regulated DEPs ([113]Figure 8A).
Figure 8.
[114]Figure 8
[115]Open in a new tab
KEGG enrichment of DEPs and hierarchical belonging of each enriched
pathway to upper level pathways. (A) Pathway classification of up
regulated DEPs. (B) Pathway classification of down regulated DEPs.
For down regulated DEPs, their pathway enrichment was mainly in
ribosome (15%), ketone metabolism and taurine metabolism ([116]Figure
8B). When evaluated with the rich factor, the enrichment of the above
DEPs exerted a different extent of influence on their corresponding
pathways. With higher rich factors, the enrichment of up regulated DEPs
probably and positively modulated alpha-linolenic acid metabolism,
tyrosine metabolism and fatty acid degradation. Meanwhile, biosynthesis
of secondary metabolites, biosynthesis of antibiotics and citrate cycle
were enriched with more up regulated DEPs but with lower rich factors
([117]Figure 9A). In contrast, the situation of down regulated DEPs was
different, in that their enrichment conferred higher rich factors for
minor pathways such as taurine and hypotaurine metabolism, and
synthesis and degradation of ketone bodies, but a lower rich factor for
a larger pathway such as ribosome, with 15 down regulated DEPs being
enriched ([118]Figure 9B).
Figure 9.
[119]Figure 9
[120]Open in a new tab
Rich-factor scatter plot of select pathway enriched with DEPs. (A) Up
regulated DEPs. (B) Down regulated DEPs. Rich factor indicating the
number ratio of differentially expressed proteins to total annotated
proteins in a pathway. Dot areas show the number of DEPs, and the
selection was based on KEGG enrichment situation and pertinence to the
metabolic blocks in the fungal response to cuminal exposure.
3.10. Integration Analysis of Transcriptome and Proteome
3.10.1. Correlations of Proteome with Transcriptome
All three biological replicates demonstrated similar replications in
the gene expression at both mRNA ([121]Figure S5A) and protein
([122]Figure S5B) levels. Among the 1485 identified proteins of the
proteome, 1362 ones were annotated. The correlation number of these
annotated proteins with annotated unigenes in transcriptome was 1305
(95.82%) ([123]Table S4) and these correlations are graphically shown
in [124]Figure S5C. Given the larger number of DEGs, we carried out a
one-by-one search for the corresponding gene in transcriptome to each
DEP. All DEPs found their encoding genes based on the matching of their
IDs ([125]Table S5), but some of these genes were not DEGs. Heat map
analysis of correlations between certain DEPs and their encoding genes
in DEGs was performed ([126]Table S6). Furthermore, among annotated
DEPs, 26 up and 24 down regulated DEPs had their encoding DEGs with the
same changing tendency, and others were not ([127]Table S7).
Noticeable up/up and down/down genes were focused on mitochondria and
ribosomes. They function in diverse types of metabolism including
citrate cycle, stress response, phospholipid metabolism, urea cycle,
transcription, fatty acid β-oxidation, electron transport, cellular
protein catabolic process, regulation of cell cycle, antibiotic
resistance, glucosidic compound degradation, energy metabolism,
translation, redox homeostasis and some cellular components biogenesis.
Interestingly, up regulated genes more enriched the stress response,
citrate cycle, phospholipid degradation and other catabolism, while
considerable genes involved in glycolysis, cell cycle and anabolism
were generally down regulated.
3.10.2. Pathway Integration of DEGs and DEPs
The DEPs were involved in 86 KEGG pathways while the DEGs were in more
than 163 KEGG pathways. To analyze the shared pathways of DEGs and
DEPs, the correlation analysis of top enriched KEGG pathways of DEPs
and DEGs were conducted. The most correlated KEGG pathways were
metabolic pathways (22), biosynthesis of secondary metabolites (13),
biosynthesis of antibiotics (12), carbon metabolism (6) and
biosynthesis of amino acids (5) ([128]Figure S6).
Among these shared pathways, the biosynthesis of antibiotics is of
interest. Moreover, ribosome, oxidative phosphorylation, terpenoid
backbone biosynthesis, fatty acid metabolism, 2-oxocarboxylic acid
metabolism, glycolysis/gluoconeogenesis, citrate cycle, steroid
metabolism, fatty acid degradation, pantothenate and CoA biosynthesis,
and valine, leucine and isoleucine degradation were well enriched
during the fungal response to cuminal exposure. Because of the large
number of genes involved in these pathways and the multifunctional
roles of these pathways, these specific pathways might be of critical
importance in demonstrating the antifungal mechanism of cuminal against
T. roseum. To further understand the importance of these KEGG pathways
during the response to cuminal, the correlation of the KEGG pathways
enriched in transcriptome and proteome was analyzed ([129]Figure 10).
Besides certain basic metabolic pathways, KEGG pathways correlated in
both transcriptome and proteome significantly embraced valine, leucine
and isoleucine degradation, biosynthesis of antibiotics, glycerolipid
metabolism, fatty acid degradation, propanoate metabolis, and
glycolysis/gluconeogenesis. Meanwhile, cell cycle, citrate cycle,
glycerophospholipid metabolism, cysteine and methionine metabolism,
pentose and glucuronate interconversions, and peroxisome, pyruvate
metabolism and steroid biosynthesis were only enriched in
transcriptome. Terpenoid backbone biosynthesis, synthesis and
degradation of ketone bodies, ribosome, and 2-oxocarboxylic acid
metabolism were only enriched in the proteome. These differences of
pathway enrichment suggest that post transcription regulation plays an
important role in the gene expression.
Figure 10.
[130]Figure 10
[131]Open in a new tab
Scatter diagram of KEGG enrichment correlation between the datasets of
proteome and transcriptome.
3.10.3. PPI upon Cuminal Exposure
KEGG pathway analysis explained how an individual protein works in the
context of a large network of various related proteins. Based on GO and
KEGG analyses of DEGs and DEPs, proteins involved in central carbon
metabolism and energy acquisition, membrane composition and drug
effluxing, stress mediation and antioxidative defenses, ribosome
formation and protein biosynthesis, and cell cycle and multiplication
were further investigated for their PPIs. These interactions are of
critical importance for the specific KEGG pathways underpinning the
above metabolic blocks and were established based on the STRING
database ([132]Figure 11).
Figure 11.
[133]Figure 11
[134]Open in a new tab
Protein–protein interaction network of T. roseum under cuminal exposure
compared with the control. The proteins encoded by DEGs functioning in
the fungal metabolism discussed in the context were selected.
Interactions are shown by the lines connecting each node based on
available evidence in the database. Interactions are integrated into
different metabolic blocks indicated by the colored circles.
Considering the lipophilicity of cuminal, the plasmalemma lipid bilayer
might first be affected by the cuminal invasion. We think this invasion
was the first biological event among the interactions of the molecule
and the fungus; this invasion might affect the constitution and
functions of the plasmalemma. To manifest how the cuminal intrusion
impacts the constitution and functions of plasmalemma, the
protein–protein interaction network of proteins encoded by DEGs
functioning in the fungal membrane composition and drug effluxing was
established ([135]Figure S7). Taking into consideration that central
carbon and energy metabolism is of pivotal importance in the vitality
and growth of any biospecies, the protein–protein interaction network
of proteins encoded by DEGs functioning in the fungal carbohydrate and
energy metabolism was shown ([136]Figure S8).
3.11. T. roseum Responses to Cuminal Stress
By a detailed analysis of specific functions of DEGs and DEPs involved
in metabolic blocks mentioned in PPI analysis, the responses of T.
roseum to cuminal exposure were further explored. In GO analysis of the
DEGs and DEPs, the most enriched biological processes were catabolic
process, oxidation-reduction process, and single-organism process; a
large part of these DEGs and DEPs belong to cell part, mitochondria,
membrane-bounded organelles and cytoplasm. Moreover, KEEG pathway
enrichments showed that central carbon metabolism, stress responses,
membrane components degradation and ribosomes were the most regulated
pathways by cuminal exposure. Accordingly, how the DEGs or DEPs
involved in these metabolic blocks correlated and how their regulation
was orchestrated were further elaborated to uncover the system response
of the fungus to cuminal.
3.11.1. Central Carbon Metabolism and Energy Acquisition
Central carbon metabolism is one of the most basic metabolic pathways
in all living cellular organisms and is considered to be the
underpinning and driving metabolism affecting cell physiology and
vitality [[137]37]. Environmental conditions can modulate the metabolic
fluxes of branches of central carbon metabolism, helping the organisms
negotiate the impacts of adverse conditions [[138]38]. Cuminal exposure
regulated the gene expression of T. roseum to adjust the fuel flux
distribution among the branches of central carbon metabolism. First,
glycolysis of the fungus slowed overall when HXK1 (Hexokinase-1), PGK1
(Phosphoglycerate kinase), and CDC19 (Pyruvate kinase 1) were down
transcripted, and the PFK2 (6-phosphofructokinase subunit beta) was up
transcripted. Consistently, the HXK1, a speed-limit key enzyme
catalyzing the conversion of glucose to glucose-6-phosphate during
glycolysis was down regulated at protein level.
The overall situation of gluconeogenesis was different from that of the
glycolysis, wherein six genes (ALD5, ACS2, THI3, ADH6, ACS1, and LAT1)
were up regulated at transcriptional level and one gene (ADH5) was up
regulated at the protein level. PCK1 (phosphoenolpyruvate
carboxykinase), a critical enzyme in gluconeogenesis, was severely down
regulated in both transcriptome and proteome, which resulted in
oxaloacetate from the upper reactions of gluconeogenesis not being
efficiently converted to phosphoenol–pyruvate and thereby having to be
metabolized through the citrate cycle. Interestingly, ACS1 and PDC6
encoding isoenzymes of ACS2 and THI3, respectively, were down
regulated, implying fine but unraveled regulation of the
gluconeogenesis. For the pentose phosphate pathway, no DEGs or DEPs
enriched its oxidative phase, but in the non-oxidative phase, PFK2
catalyzing the one-way reaction of β-D-fructose-6P to
β-D-fructose-1,6P2 was up transcripted, thereby supplying the
non-oxidative phase with the D-glyceraldehyde-3P. Moreover, in the
non-oxidative phase, two genes (PGM2 and TAL1) and one gene TKL1 were
down regulated at transcriptional and translational level,
respectively. Nonetheless, owing to the supplement of metabolites to
the non-oxidative phase from other linked metabolisms, such as pentose
and glucuronate interconversions, and other carbohydarate metabolisms,
the effects of the down regulation of certain non-oxidative phase
enzymes on the generation of reducing power from pentose phosphate
pathway might be compensated for somewhat, so the cells still possessed
weak anti-oxidative capacity.
Cuminal exposure regulated the citrate cycle more positively. A total
of seven genes (KGD1, LSC2, IDP3, SDH1, CIT2, LAT1, and IDP1) were up
regulated at the transcript level, and among them, CIT2, IDP1 and KGD1
encode the key enzymes of the cycle citrate synthase, isocitrate
dehydrogenase, and 2-oxoglutarate dehydrogenase, respectively. However,
LSC1, CIT1 and MDH2 were down regulated. The down regulation of LSC1
and CIT1 might be offset by the significant overexpression of their
corresponding family gene LSC2 and CIT2. Although the MDH2 was down
regulated, its isoenzyme MDH1 was overexpressed in proteome.
Interestingly, one critical enzyme of the citrate cycle, the
2-ketoglutarate dehydrogenase (KGD1) was highly (2.68-fold)
overexpressed in proteome too. Furthermore, no down regulated proteins
enriched the citrate cycle. Overall, metabolism of the citrate cycle
was enhanced largely due to the elevation of β-oxidation, val, leu and
Ile degradation, and other amino acid degradation and these pathways
were significantly enriched in both omic datasets.
The regulation of β-oxidation seems more straight and positive than
other metabolisms. The enzyme 3-ketoacyl-CoA thiolase (POT1) was up
regulated at both mRNA (5.52-fold) and protein (1.57-fold) levels, and
this enzyme catalyzes the releasing of acetyl-CoA from the β-oxidation
and has a key role in fatty acid degradation [[139]39]. Long-chain
acyl-CoA synthetase (FAA1), an enzyme of the ligase family, was
extremely (373.42-fold) up regulated at mRNA level and the enzyme
activates the breakdown of complex fatty acids and is also involved in
the esterification, with concomitant transport, of exogenous long-chain
fatty acids into metabolically active CoA thioesters for subsequent
degradation. Additionally, two genes (DIT2 and ALD5) functioning in
affiliated metabolism of the β-oxidation were up regulated too, and
they degrade alkanes, 1-alcohols and aldehydes before β-oxidation. No
down regulated DEGs enriched the β-oxidation pathway. Furthermore,
alcohol dehydrogenase (ADH5) was overexpressed at the protein level,
enhancing the transferring of 1-alcohols to aldehydes and preparing
more fuels to β-oxidation. Altogether, the elevated β-oxidation
supplied more acetyl-CoA molecules to citrate cycle, and thus the
fungus obtained a compensation for the down regulation of glycolysis.
Given the just mentioned situations, the productivity of the
glycolysis, even down regulated in general, may supply some
intermediates to the pentose phosphate pathway to ensure subsistence
reducing power during the fighting of stress.
In eukaryotes, oxidative phosphorylation is carried out by a series of
protein complexes composing the electron transport chains within the
inner membrane of mitochondria. Cuminal exposure did not regulate the
genes encoding the subunit of complex I (NADH-coenzyme Q
oxidoreductase). Of complex II (Succinate-Q oxidoreductase), succinate
dehydrogenase (ubiquinone) flavoprotein subunit 1 (SDH1) and subunit 2
(YJL045W) were down regulated at mRNA level. For complex III
(Q-cytochrome c oxidoreductase), ubiquinol–cytochrome c reductase core
subunit 2 (QCR2) was down transcripted. Consistently, of complex IV
(Cytochrome c oxidase), cytochrome c oxidase assembly protein subunit
11 (COX11) was highly down regulated at mRNA level, and cytochrome c
oxidase subunit 4 (COX5A) was down at the protein level. More
importantly, mitochondrial ATP synthase subunit 9 (OLI1) of complex V
(ATP synthase) was down regulated, though the β-subunit of the F-type
ATPase (ATP2) was up regulated. Supply of phosphate (Pi) from PPi
hydrolysis is necessary for the well performance of complex V, but the
gene of inorganic pyrophosphatase, mitochondrial (PPA2) was down
regulated at the mRNA level, which may weaken the complex V.
3.11.2. Membrane Constitution and Drug Effluxing
Plasmalemma plays a pivotal role in maintaining the sound function of
most other cellular components. Membrane component reconstitution could
adapt the cells to the environmental changes. Glycerophospholipids
compose the framework of the plasmalemma and their metabolism
essentially is a restructuring of the cellular membranes [[140]40].
Cuminal exposure up regulated the transcription of certain genes
functionally involved in membrane lipids metabolism. Among the proteins
they code, choline kinase (CKI1) transfers the choline into
phosphor–choline, and this activated choline molecule is a precursor of
phosphatidylcholine. CDP-diacylglycerol-inositol
3-phosphatidyltransferase (PIS1) catalyzes CDP-diacylglycerol to
phosphatidyl-1D-myo-inositol involving in inositol phosphate metabolism
and GPI-anchor biosynthesis. 1-Acylglycerone phosphate reductase (AYR1)
and lysophosphatidate acyltransferase (SLC1) catalyze successive
bio-reactions to transfer 1-acyl-glycerone to 1,
2-diacyl-sn-glycerol-3P. Diacylglycerol kinase (DGK1) converts the 1,
2-diacyl-sn-glycerol to 1, 2-diacyl-sn-glycerol-3P that then
participates in the inositol phosphate metabolism or is further
converted to phosphatidylethanolamine. In fungal cells,
lysophospholipid acyltransferase (ALE1) catalyzes the
1-acyl-sn-glycero-3-phosphoethanolamine to phosphatidylethanolamine,
and ethanolaminephosphotransferase (EPT1) catalyzes the integration of
CDP-ethanolamine and 1, 2-diacyl-sn-glycerol to form
phosphatidylethanolamine. The phosphatidylethanolamine
N-methyltransferase (CHO2) converts phosphatidylethanolamine to
monomethyl–phosphatidylethanolamine, an intermediate to synthesize the
phosphatidylcholine. Altogether, the up regulated glycerophospholipid
metabolism genes could elevate the biosynthesis of phosphatidylcholine
and inositol phosphate metabolism. The down regulation of cardiolipin
synthase (CRD1) may limit the conversion of CDP-diacyl-glycerol to
cardiolipin and thus more CDP-diacyl-glycerol molecules could be
metabolized into phosphatidylethanolamine. Concordantly, in the
proteome, phosphatidylserine decarboxylase (PSD2) that catalyzes
phosphatidyl-L-serine to phosphatidylethanolamine was up regulated. In
contrast, no down regulated DEPs enriched the glycerophospholipid
metabolism.
Sterols are hallmarks of the eukaryotic membranes and reconstitution of
these components modulates the function of the membranes. Four genes
(ERG25, ERG9, ERG6, and ERG5) involved in sterol biosynthesis were down
regulated. De novo biosynthesis of steroids derived its precursors from
terpenoid backbone biosynthesis. Farnesyl-diphosphate
farnesyltransferase (ERG9) catalyzes the producing of squalene, and
highly down regulation of the enzyme suggests less squalene generated,
which therefore limits the biosynthesis of the subsequent intermediates
of the pathway. The down regulation of methylsterol monooxygenase
(ERG25) may limit the biosynthesis of zymosterol, an important
precursor of ergosterol biosynthesis, subsequently catalyzed by sterol
24-C-methyltransferase (ERG6) and sterol 22-desaturase (ERG5), both of
which were down transcripted, and ergosterol is the precursor of
vitamin D2. Although certain genes coding enzymes catalyze the
downstream metabolism since ergosterol were up regulated at transcript
level, this up regulation does not necessarily mean an elevation of the
sterol biosynthesis, but rather, a more sophisticated and unraveled
regulation of steroids metabolism under stress.
Facing stresses, microbial cells could re-modulate the ratio of
saturated to unsaturated fatty acids to survive the adverse conditions
[[141]41]. Cuminal exposure down regulated two genes coding acyl-CoA
oxidase (POX1) and elongation of fatty acids protein 2 (ELO2) at mRNA
level, both of which are involved in the biosynthesis of
polyunsaturated fatty acids especially linolenic acid, arachidonic
acid, icosapentaenoic acid and docosahexaenoic acid. Nonetheless,
3-ketoacyl-CoA thiolase (POT1, 5.52-fold in transcriptome; 1.57-fold in
proteome) was up regulated in both omic datasets, but the enzyme
elevation herein strengthens the β-oxidation. These adjustments of the
relevant gene expression suggested that the unsaturated fatty acids
biosynthesis was limited and that breakdown of lipid fatty acids via
β-oxidation increased. The gene (FAA1) coding the
long-chain-fatty-acid-CoA ligase 1, an important key enzyme of fatty
acid degradation, was highly overexpressed (373.42-fold) at mRNA level.
Another two genes, DIT2 and ALD, encoded enzymes involved in the
degradation of long chain alcohols and aldehydes were up regulated too.
Additionally, up regulation of alcohol dehydrogenase 5 (ADH5) at the
protein level could also facilitate the catabolism of alcohols from
lipid degradation.
Cuminal treatment up regulated four genes (PDR5, ATM1, SNQ2 and STE6)
relating to drug efflux pumps functions. Among the proteins coded by
these four genes, pleiotropic ABC efflux transporter of multiple drugs
(PDR5) is responsible for active effluxing of weakly charged organic
compounds, and confers resistance to numerous chemicals including
antiseptics, antibiotics, herbicides, mycotoxins and other antifungals
[[142]42]. Consistently, the PDR5 was overexpressed (4.51-fold) at
protein level. Iron–sulfur clusters transporter ATM1, in concert with
glutathione, functions in the export of a substrate required for
cytosolic-nuclear iron–sulfur protein biogenesis and cellular iron
regulation, protects the cell against oxidative stress, and plays a
role in vacuolar functions in isolating and exporting materials that
might be harmful or a threat to the cell [[143]43]. ABC export permease
SNQ2 acts as pleiotropic drug resistance transporter and confers cells
the capability of exporting drugs [[144]44]. Alpha-factor-transporting
ATPase (STE6) is a full-size ABC-B transporter distributed in all
organisms and is involved in multidrug resistance [[145]45].
3.11.3. Stress Mediation and Antioxidative Defenses
Environmental toxic agents can induce the increase of ROS level
[[146]46], resulting in oxidative stress (OS) if the ROS are over
accumulated in the organisms. For survival upon cuminal exposure, the
fungus drew on antioxidant arsenals to minimize the detrimental effects
of oxidative stress, and several genes involved in the peroxisome
pathway were modulated. Peroxisomal catalase A (CTA1), occurring in
almost all aerobic organisms and being able to protect cells from the
toxic effects of hydrogen peroxide during the response to reactive
oxygen species [[147]47], was overexpressed. Two genes (IDP1 and IDP3)
encoding isocitrate dehydrogenase [NADP] (IDH) were also overexpressed.
IDH is involved in the NADPH regeneration and hence cellular processes
depending on NADPH, and IDH suppresses intracellular and mitochondrial
ROS level [[148]48]. Highly (39.48-fold) expressed SYM1 encodes protein
Mpv17l involved in cellular response to stress and required to maintain
mitochondrial DNA integrity [[149]49]. Mpv17l is also involved in
peroxisomal reactive oxygen species metabolism and protects cells
against mitochondrial oxidative stress by activation of Omi/HtrA2
protease [[150]50]. SOD2 encoding superoxide dismutase [Mn] of
mitochondria was highly transcripted (33.28-fold), and this protein
scavenges toxic superoxide anion radicals normally produced within the
cells [[151]51].
Cuminal exposure positively regulated several genes involved in
mitophagy. Mitophagy, a selective degradation of mitochondria by
autophagy, belongs to the oxidative defenses and promoters of life
extension when correctly regulated [[152]52]. Up transcripted cell wall
integrity and stress response component 3 (WSC3, 2.26-fold) is a cell
surface stress sensor detecting mitophagy-inducing stimuli and
activating the Hog1 and Slt2 signaling pathways, facilitates the
formation of the autophagosome surrounding the mitochondria and its
eventual fusion with vacuoles for mitochondrial degradation [[153]53],
and also elevates the pleiotropic drug resistance [[154]54]. With an
ATP-independent isopeptidase activity, ubiquitin carboxyl-terminal
hydrolase 3 (UBP3, 5.64-fold) was up transcripted, and the enzymeis
required for efficient stress granule assembly in Saccharomyces
cerevisiae [[155]55]. Stress granules have long been proposed to
function in protecting RNAs from stress conditions [[156]56]. Up
transcripted SLG1 plays a role in regulating the entering or exiting
the cell cycle and has a functional link between mitochondrial RNA
editing and responses to abiotic stress [[157]57]. Casein kinase II
subunit alpha (CKA1) was up regulated at mRNA level and is an enhancing
factor in abiotic stress signaling via modulating the expression of
some molecular elements in retrograde signaling [[158]58].
Transcriptically up regulated UBP3-associated protein BRE5 (BRE5) is
involved in the formation of stress granules appearing when cells are
under stress [[159]59].
Consistently, down regulation of the following genes demonstrates that
the fungal cells experienced an increased stress and responded to it as
a price to survival. Catalytic mitochondrial inner membrane i-AAA
protease super-complex subunit YME1 (YME1) is required for
mitochondrial inner membrane protein turnover and is important to
maintain the integrity of the mitochondrial compartment. At the protein
level, this protein was up regulated 1.57-fold too. The YME1 is
essential both for the degradation of unassembled subunit 2 of
cytochrome c oxidase and for efficient assembly of mitochondrial
respiratory chain [[160]60]. Autophagy-related protein 11 (ATG11)
recruits mitochondria for their selective degradation during starvation
through its interaction with autophagy-related protein 32 and plays a
significant role in life span extension [[161]61]. Mitogen-activated
protein kinase HOG1 (HOG1) plays a crucial role in the response to
various environmental stresses, and mutations in HOG1 render an
organism more sensitive to agents producing reactive oxygen species,
such as oxidants and UV light [[162]62]. The SSK1 product may fulfil
regulatory roles in signaling pathways involving a HOG1 MAP kinase
during ROS tolerance, osmotic resistance, fungicide sensitivity and
fungal virulence [[163]63]. Vacuolar protein sorting-associated protein
1 (VPS1) and Atg8 cooperatively participate in vacuolar function,
thereby contributing to oxidative stress resistance [[164]64]. Loss of
the mitochondrial distribution and morphology protein 38 (MDM38) in
yeast mitochondria results in a variety of phenotypic effects including
reduced content of respiratory chain complexes, altered mitochondrial
morphology, and loss of mitochondrial K(+)/H(+) exchange activity
[[165]65].
3.11.4. Ribosome Formation and Protein Biosynthesis
Cells typically respond quickly to stress and alter their metabolism to
compensate for their stress. The nucleolus senses stress and is a
central hub for coordinating the stress response, which is fulfilled by
rapid production of small and large ribosome subunits, a process that
must be highly regulated to achieve proper cellular proliferation and
cell growth [[166]66]. Ribosome biogenesis occurs sequentially in the
nucleolus, the nucleoplasm and the cytoplasm. It involves the
transcription and processing of pre-ribosomal RNAs, their proper
folding and assembly with ribosomal proteins, and the transport of the
resulting pre-ribosomal particles to the cytoplasm where the final
maturation events take place [[167]67]. Several DEGs enriched the
ribosome biogenesis. The seven up regulated DEGs (EMG1, UTP14, UTP10,
CKA1, FCF1, UTP22 and PWP2) are all involved in the formation of 90S
pre-ribosome in the nucleolus. Casein kinase II subunit alpha (CKA1)
and U3 small nucleolar RNA-associated protein 22 (UTP22) are factors of
UTP-C complex; U3 small nucleolar RNA-associated protein 10 (UTP10) is
a factor of t-UTP complex; periodic tryptophan protein 2 (PWP2) is a
factor of UTP-B complex. These three complexes participate in the
formation of 90S pre-ribosome together with U3 small nucleolar
RNA-associated protein 24 (FCF1) and U3 small nucleolar RNA-associated
protein 14 (UTP14) as well as 18S rRNA pseudouridine methyltransferase
(EMG1). The cleavages of 90S pre-ribosome creates pre-40S and pre-60S
ribosomal particles. Then, the both particles are transported out of
the nucleolus and into the cytoplasm. Once in the cytoplasm, the
pre-60S ribosomal particle further undergoes processing to be
functional. To this end, the maturation requires many biogenesis
factors. But after cuminal treatment, the 18S rRNA pseudouridine
methyltransferase (MDN1), large subunit GTPase 1 (LSG1) and ribosome
assembly protein 1 (RIA1) were down regulated. The enrichment of these
down regulated DEGs probably made the 60S ribosome subunit not
workable, but the precise mechanism remains unclear since pathway of
the 60S subunit cytoplasmic maturation is not yet well understood
[[168]68].
From transcription to RNA processing and translation, expression
regulation of coding genes is controlled at multiple levels. mRNA
maturation and translation are post-transcriptional regulatory
mechanisms underpinning the genome’s coding capacity in modifying
protein function, stability, localization and expression levels
[[169]69]. Several genes involved in mRNA maturation and translation
were regulated by cuminal exposure. Nuclear cap-binding protein subunit
1(STO1) binds co-transcriptionally to the 5’-cap of pre-mRNAs and is
involved in the degradation of nuclear mRNAs. FUN12 encoding the
translation initiation factor eIF5B was up regulated after cuminal
exposure; eIF5B may be one of the targets among the translation
components affected by redox [[170]70]. As to those down regulated DEGs
involved in the mRNA processing and translation, they are generally
functioning in the translation initiation factors and exon-junction
complex. Eukaryotic translation initiation factor 1A (TIF11) is
required for maximal rate of protein biosynthesis and enhances ribosome
dissociation into subunits and stabilizes the binding of the initiator
Met-tRNA(I) to 40 S ribosomal subunits [[171]71]. Polyadenylate–binding
protein (PAB1) appears to be an important mediator of the multiple
roles of the poly(A) tail in mRNA biogenesis, stability and translation
[[172]72]. ATP-dependent RNA helicase (FAL1) is an element of
exon-junction complex and responsible for exon splicing. ATP-dependent
helicase (NAM7) is required for rapid turnover of mRNAs containing a
premature translational termination codon [[173]73]. Eukaryotic
translation initiation factor 2 subunit gamma (GCD11) functions in the
early steps of protein synthesis by forming a ternary complex with GTP
and initiator tRNA, and the yeast eIF2γ mutation impairs translation
start codon selection and thus vitiates translation initiation
[[174]74]. Down regulation of these critical genes implies that cuminal
exposure impaired the biosynthesis of proteins.
3.11.5. Cell Cycle and Multiplication
T. roseum reproduces asexually through the formation of conidia with no
sexual state [[175]75]. Cuminal exposure altered the transcripts of
several genes of the fungus involved in cell cycle pathways, and some
of them were up regulated. Among these elevated genes, cell division
control protein 45 (CDC45) is required for initiation of chromosomal
DNA replication and also has a role in minichromosome maintenance
[[176]76]. Guanine nucleotide exchange factor LTE1 (LTE1) is a GDP-GTP
exchange factor for GTP-binding protein involved in the termination of
M phase and functions in the mitotic exit network. LTE1 as a signal
promotes exiting from mitosis by multiple mechanisms [[177]77].
Structural maintenance of chromosomes protein 4 (SMC4) is a central
component of the condensin complex required for conversion of
interphase chromatin into mitotic-like condense chromosomes [[178]78].
F-box protein MET30 (MET30) regulates several aspects of the cell
cycle, including G (1)-specific transcription, initiation of DNA
replication, and M phase progression [[179]79]. The elevation of such
genes suggests that the fungus is on the alert for the possibility of
cuminal-caused destruction of chromosomes.
In contrast, more genes involved in cell cycle were down transcripted.
multi-functional protein phosphatase PP2A regulatory subunit A (TPD3)
affects transcription, cell cycle progression, and cellular
morphogenesis [[180]80]. RING-box protein HRT1 (HRT1) targets Pol II
for proteasomal degradation in DNA-damaged cells and thus affects cell
division [[181]81]. The anaphase promoting complex/cyclosome activator
protein CDH1 (CDH1) regulates the ubiquitin ligase activity and
substrate specificity of the anaphase promoting complex/cyclosome and
is required for exit from mitosis, cytokinesis and formation of
prereplicative complexes in G1. Cell cycle serine/threonine-protein
kinase (CDC5) is involved in mitotic exit, and functions in preserving
of genome integrity [[182]82]. Serine/threonine protein kinase (KCC4)
plays a role in cell wall synthesis and is involved in budding cell bud
growth [[183]83]. Structural maintenance of chromosomes protein 2
(SMC2) is the central component of the condensin complex required for
conversion of interphase chromatin into mitotic-like condense
chromosomes [[184]84]. The product of DBF4 is involved in cell cycle
regulation of pre-mitotic chromosome replication and in chromosome
segregation. DNA replication licensing factors MCM2 (MCM2), MCM4
(MCM4), and MCM7 (MCM7) act as a component of the MCM complex, which is
the putative replicative helicase essential for ’once per cell cycle’
DNA replication initiation and elongation in eukaryotic cells
[[185]85]. Cyclin-dependent kinase 1 (CDC28) is essential for the
initiation, the controlling event, of the cell cycle [[186]86]. Mitotic
check point protein (BUB2) is a part of a checkpoint monitoring spindle
integrity and preventing premature exit from mitosis. Origin
recognition complex subunit 1 (ORC1) binds origins of replication and
has a role in chromosomal replication. Serine/threonine-protein
phosphatase PP2A-1 catalytic subunit (PPH21) is involved in the control
of G1/S transition of mitotic cell cycle [[187]87].
Serine/threonine-protein kinase CHK1 (CHK1) is required for
checkpoint-mediated cell cycle arrest and for activation of DNA repair
in the presence of DNA damage or un-replicated DNA [[188]88]. The down
expression of these cell cycle related genes denotes the arresting of
fungal cell cycle, precipitating failure of cellular multiplication.
In proteome, no up regulated protein involved in cell cycle was
detected while certain down regulated proteins enriched this cell
process. Minichromosome maintenance protein 5 (MCM5) acts as component
of a putative replicative helicase essential for ’once per cell cycle’
DNA replication initiation and elongation in eukaryotic cells
[[189]85]. Checkpoint serine/threonine-protein kinase (BUB1) is
involved in cell cycle checkpoint enforcement and was the most down
modulated protein, decreasing nearly 7-fold, in this cellular process.
Structural maintenance of chromosomes protein 1 (SMC1) functions in
chromosome cohesion during cell cycle and DNA repair. The enrichment of
such proteins corroborated the occurrence of cell cycle arresting.
Additionally, purine salvage is a complex pathway allowing for a
correct balance between adenylic and guanylic derivatives; a decrease
of the guanylic nucleotide pool connotes cell shifting from
proliferation to quiescence. Guanine deaminase, a critical enzyme in
purine salvage, was elevated both at transcriptional (GUD1)
(12.86-fold) and translational (1.78-fold) levels. This enzyme
catalyzes the hydrolytic deamination of guanine, producing xanthine and
ammonia [[190]89]. Up transcripted 3’,5’-cyclic-nucleotide
phosphodiesterase (PDE1) catalyzes nucleoside 3’,5’-cyclic phosphate to
nucleoside 5’-phosphate [[191]90], suggesting a decrease of the cAMP
level. Regulation of intracellular levels of cyclic nucleotides is
among the mechanisms involved in cell cycle progression and is of
critical importance for cell survival [[192]91].
4. Discussion
The essential oil of C. cyminum has been extensively explored for its
antioxidant and antimicrobial activities [[193]92,[194]93], but most of
the investigations focused only on the bioactivities of the essential
oil and have seldom considered what is the most responsible component
for its specific bioactivity. Although synergy was a popular account of
the essential oil bioactivities [[195]94,[196]95], overemphasizing
synergy is not useful for the elucidation of the mode of these
bioactivities and consequently, for the development of more effective
remedies. Since the preciousness of natural-derived essential oils
makes it financially unfeasible to practically use them to achieve the
desired effects, the investigation of the function mechanisms of their
bioactive components is of practical significance, so that some low
cost alternatives can be developed. Our earlier research has shown that
cuminal is the most predominant component of the C. cyminum essential
oil and acidolysis pretreatment can elevate the contents of cuminal and
the antifungal activity of the essential oil [[197]26]. Indeed, the
antifungal activity of the individual cuminal was twice as strong as
that of the essential oil itself, suggesting that intensive examination
of the function mode of cuminal against fungal growth will be conducive
to the interpretation of the antifungal mechanism of cumin essential
oil.
As the predominant component of the cumin essential oil, lipophilic
cuminal molecules should have strong affinity to cytoplasm membrane;
thus, cuminal exposure elicited the fungal responses from the membrane
first. When intruding onto the lipid bilayer of cytoplasm membrane,
cuminal damaged the polarity of the cytoplasm membrane. Membrane
depolarization is associated with the membrane fluidity decrease and
compromised functionality [[198]96]. In the present investigation,
cuminal exposure rendered the fungal transcription of genes functioning
in membrane lipids metabolism up regulated. These up regulated genes
mainly collaborate in the biosynthesis of phosphatidylcholines,
inositol phosphates and phosphatidylethanolamines. In contrast, the
down regulation of cardiolipin synthase suggests the limited
cardiolipin biosynthesis. Both transcriptome and proteome
investigations showed the elevation of the biosynthesis of
phosphatidylethanolamines, but the reason for such an elevation was not
reported. Additionally, a number of eukaryotic host defense peptides
such as plant cyclotides use phosphatidylethanolamines as a receptor to
promote their antimicrobial activities [[199]97]. These changes of
membrane lipid biosynthesis may alter the ratios of various lipid
species of the membrane; the resulting lipid profile changes might
impact the interactions among these molecules and so induce membrane
disorder [[200]98]. Fungal plasmalemma is a universal target explored
extensively for the development of antifungal agents. This strategy has
been proved fruitful by the pronounced success of antifungal drugs such
as azoles and polyenes [[201]99]. Consistent with the down regulation
of most of membrane phospholipid synthesis was the overall trend of
ergosterol biosynthesis of the membrane. Ergosterol is the primary
sterol in fungal membranes and presumably contributes to membrane
fluidity and function [[202]100,[203]101]. The fungus responded to
cuminal exposure with the down regulation of the ergosterol
biosynthesis genes (ERG25, ERG9, ERG6, and ERG5), and this regulation
was consistent with the decrease in ergosterol content of the cellular
membrane (data not published). By increasing the permeability of
membrane, modulating the activity of membrane-bound enzymes in the
plasmalemma and mitochondria, stimulating uncoordinated chitin
synthesis or interfering with fatty acid synthesis, a deficiency in
ergosterol affects fungal viability and growth [[204]102]. Furthermore,
and interestingly, cuminal exposure strengthened the biosynthesis of
polyunsaturated fatty acids. This positive adaptation of the fungus to
the adverse conditions is common in other stress threatened microbes
[[205]103,[206]104]; the up regulated desaturation is a cellular
response to environmental stresses, protecting cells from toxic oxygen
species and other detrimental factors [[207]105].
ABC transporter proteins are crucial for pleiotropic drug resistance,
stress response and cellular detoxification of fungi [[208]106].
Cuminal exposure elevated the gene expression of these kinds of
proteins in both transcriptome and proteome. This elevation might be
instinct protection against adversities, but this endeavor seems futile
in conquering the overwhelming intrusion of cuminal. Accordingly,
cuminal intrusion into the cytoplasmic membrane begot further cellular
defensive responses and resulted in dysfunction of the membrane.
Another important consequence of cuminal exposure was the occurrence of
oxidative stress (OS) upon the fungus because environmental toxic
agents can induce the level escalation of ROS in aerobic organisms
surrounded by such agents [[209]46]. OS results when production of ROS
exceeds the removing of these toxic species by cellular antioxidant
systems, and some important systems of such involve special cellular
organelles and antioxidant enzymes including superoxide dismutase,
catalase, and peroxidases, which detoxify the cells of harmful ROS,
thereby reducing damage to the cells [[210]107]. Peroxisomes are highly
dynamic and metabolically active organelle playing a critical role in
regulating cell perception and fast responses to environmental cues of
stresses [[211]108]. Accumulating evidence indicates that peroxisomes
are of primary importance during the maintenance of the cellular
oxidative homeostasis [[212]109], and, in the present investigation, up
regulation of certain key genes functioning in oxidative defense might
enhance the fungal tolerance to cuminal. The failure of sustaining
supply of reducing power by the collapsed carbohydrate metabolism,
meanwhile, aborted such adaptive attempt in facing the persistent
oxidative attacks of cuminal exposure.
Autophagy, a catabolic process for recycling cellular components and
damaged organelles, possibly occurs when cells are under diverse stress
conditions. Acting as the universal and converging stress directly from
some oxidative chemicals or derivatively from some adversities such as
starvation or poisoning, OS induces sustaining autophagy [[213]110].
When it properly occurs, mitophagy, a selective autophagy of
mitochondria during the maintenance of cell homeostasis, can mitigate
the oxidative stress and protect cells from noxious ROS
[[214]111,[215]112]. The process of mitophagy plays multimodal roles in
the survival of cells. Benign mitophagy renders the cell adaptive to
certain levels of stress, but under lasting or overwhelming stress,
abnormal mitophagy takes place and compromises the cells viability
[[216]113,[217]114]. Mitochondria play a pivotal role in the energy
acquisition of eukaryotes and too great a loss of the crucial organelle
destroys the energy metabolism of an organism. The escalation of
mitophagy of T. roseum upon cuminal exposure might be one aspect of
mechanism underpinning the drug inhibition.
As mentioned, cuminal-instigated disturbance in the cytoplasmic
membrane component profiles would trigger the dysfunction of the
membrane and destroy the nutrient uptake, thus causing the nutrition
deprivation to the fungus. This deprivation diminished the central
carbon metabolism flux of the fungus. Integrated transcritome and
proteome analysis showed the glycolysis was generally down regulated
particularly due to the low expression of the HXK1 (encoding
hexokinase-1) and the CDC19 (pyruvate kinase 1), since both of the
enzymes are speed-limit ones to the glycolysis. The down regulation
indicates the fungal cell carbohydrate starvation resulting from the
dysfunction of cytoplasmic membrane. For surviving, the cells have to
draw on the other interconnected metabolites to compensate for this
carbohydrate deprivation. Gluconeogenesis is a typical way to replenish
the carbohydrates and is sensitive to glucose deficiency [[218]115]. Up
regulation of gluconeogenesis could transiently refuel the cells while
consuming precursor metabolites derived from other metabolism and
finally resulting in the comprehensive starvation of the cells. Long
term starvation renders the wild type Neurospora crassa more sensitive
to heat shock and oxidative stress and brings about lethality to the
fungus [[219]116]. In the present investigation, gluconeogenesis
experienced more sophisticated regulation. Although certain genes were
up regulated at transcription level, the down regulation of PCK1 at
both mRNA and protein levels indicates that more intermediate pyruvate
might be diverted to the citrate cycle, which further proved the
glucose starvation of the fungus. The overall down regulation of the
oxidative phase of pentose phosphate pathway indicated that NADPH
production declined. Lack of the NADPH crippled the anabolic reactions
for the biosynthesis of nucleic acids and lipids and weakened the
oxidative stress defense, thereby reducing the growth or viability of
the fungus [[220]117].
Opposite to the regulation of glycolysis was the elevation of the
citrate cycle under the treatment of cuminal. The overall positive
modulation of citrate cycle of the T. roseum seems at odds with the
down regulation of the glycolysis, whereas, in fact, the active
β-oxidation and strengthened degradation of val, leu, Ile and other
amino acids could fuel the citrate cycle with acetyl CoA, which
accounts for the elevation of citrate cycle. Furthermore, carbohydrate
deprivation promotes the lipid droplets-mitochondria interaction and
lipid droplets efficiently supply fatty acids for mitochondrial
β-oxidation [[221]118]. The products of β-oxidation, especially
acetyl-CoA, feed the citrate cycle of central carbon metabolic pathways
[[222]119]. Moreover, acute carbohydrate deprivation induces autophagy
and elevates amino acid catabolism, which helps maintain transient
homeostasis of cells in nutrient scarce conditions to facilitate their
viability [[223]120].
A universal consequence of the nutrient deprivation was the failure of
bioenergetic system of cells. Cuminal exposure of T. roseum down
regulated certain components of respiratory chain complex II, III, IV
and V, and thus crippled the oxidative phosphorylation of the fungus.
These modulations on respiratory chains manifested mitochondrial stress
resulting in reduced electron transport efficiency and cellular energy
deficiency [[224]121]. Also, decreases in respiratory chain complex
activities are thought to be associated with oxidant/antioxidant
imbalance and induce cellular degeneration [[225]122]. Considering that
mitochondria are the primary source of ROS, less efficient respiratory
chains means that more ROS are generated, which exacerbates the
oxidative stress of cells [[226]123]. Cumulatively, these deleterious
effects could eventually cause cellular oxidative damage and beget the
loss of cell viability.
Under the deprivation of carbohydrate and resultant energy deficiency,
the biosynthesis of other cellular components might be inevitably
vulnerable. Cuminal exposure effectuated diminished central carbon
metabolism and incapacitated the supplies of enough precursors, energy,
and reducing powers for the anabolism of essential components for the
cell survival and proliferation. In present investigation, an active
modulation of ribosomal formation was observed. More up regulated genes
enriched the pre-ribosome formation, and this might be an attempting
protection against the abiotic stress, but very little is known about
this sophisticated process. Nonetheless, several genes, such as MDN1,
LSG1 and RIA1, being pivotal to ribosome maturation, were down
regulated, which denotes the failure of providing sufficient
efficacious translational apparatus. Furthermore, a notable amount of
genes functioning in the translation initiation and mRNA maturation
were down regulated, further impairing the protein biosynthesis.
Eukaryotic cells have developed sophisticated systems to constantly
monitor changes in the extracellular environment and to orchestrate
proper cellular responses so as to accomplish stress adaptation. To
maximize survival, cells delay cell-cycle progression in response to
environmental insults [[227]124]. Activation of stress responses can
induce diverse physiological changes, including modulation of cell
cycle progression [[228]125] and excessive stress on replication
occasions mitotic cell death [[229]126,[230]127]. In line with the
above statements, after cuminal treatment, an overall down regulation
of the genes functioning in DAN replication, cell cycle and cell
proliferation was observed. This was not surprising, because the energy
deprivation of the fungus begot a deficiency of the nucleotides, the
raw materials of nucleic acid biosynthesis, which precipitates stress
on cell replication [[231]128] of the fungus. Intense stress instigates
the transition of cells from growth to quiescence, which is often
accompanied with cell cycle modulation, housekeeping function
down-regulation, and fierce metabolism changes, all as strained
protections against stress [[232]129]. The deactivation of cell
replication involves stepwise physiological changes with an
intermediate state of being incapable of initiating replicative
processes but still capable of metabolism, and this loss of replication
competency eventually leads to cell death [[233]130]. All the fungal
responses, aforementioned, to cuminal exposure could be delineated on a
logical diagram of proposed mode of action to conclude the antifungal
mechanism of cuminal against T. roseum ([234]Figure 12).
Figure 12.
[235]Figure 12
[236]Open in a new tab
Proposed mode of action of cuminal against T. roseum. Red color in the
diagram shows up regulation of genes or overall elevation of the
indicated metabolic blocks while green color indicates down regulation
of genes or overall suppression of a metabolic bock. The metabolism
blocks that were deeply analyzed in the text are circled with red or
green dash lines.
5. Conclusions
Cuminal inhibited the growth of T. roseum in vitro and in vivo.
Electron microscopic observations disclosed the inhibition is related
to the degenerating of cellular ultrastructure, especially the
plasmalemma. Cuminal exposure regulated T. roseum gene expression at
both transcriptome and proteome levels. Totally, omics analysis
determined 2825 differentially expressed transcripts (1516 up and 1309
down) and 225 differentially expressed proteins (90 up and 135 down).
Overall, notable parts of these DEGs functionally enriched subcellular
loci of membrane system and cytosol, along with ribosomes,
mitochondria, and peroxisomes. In line with the locality analysis,
carbohydrate and lipids metabolism, redox homeostasis, and asexual
reproductive were among the most enriched GO terms in biological
annotation of these DEGs. The up regulated genes more enriched the
lipids degradation and antioxidant responses, and the down regulated
ones, in contrast, enriched the carbohydrate catabolism, energy
acquisition, cellular reproduction, and ribosomal functions. Moreover,
the KEGG pathway enrichments of the DEGs embraced elevated lipids and
amino acids degradation, ATP-binding cassette transporters, membrane
reconstitution, mRNA surveillance pathway, and peroxisome, along with
the diminished secondary metabolite biosynthesis, cell cycle, and
glycolysis/gluconeogenesis. Furthermore, integrated omics analysis
demonstrates that cuminal first impaired the functional integrity of
cytoplasmic membrane and triggered the reconstitution and dysfunction
of membrane resulting in handicapped nutrients procurement of the
cells. Consequently, cell starvation occurred, and cellular
carbohydrate metabolism was limited, and the cells might have to depend
more on the degradation of their own components in response to the
stress. Additionally, these predicaments occasioned oxidative stress,
which, in collaboration with the starvation, damaged certain critical
organelles such as mitochondria. Such degeneration together with energy
deficiency suppressed the biosynthesis of essential proteins and
paralyzed the cell multiplication.
Acknowledgments