Exploring low-dose gamma radiation effects on monoterpene biosynthesis in Thymus vulgaris: insights into plant defense mechanisms

Abstract

Thymus vulgaris, commonly known as thyme, is a plant renowned for producing monoterpenes. This study aimed to understand the effects of low-dose gamma radiation, specifically in the range of 1–5 Gy, on various traits of Thymus vulgaris, providing context on its importance in agricultural and medicinal applications. The research explored morpho-physiological, biochemical, and gene-expression responses in thyme plants under no gamma- and gamma-ray exposure conditions. The study revealed complex relationships between gamma-ray doses and plant characteristics. In particular, shoot and root lengths initially increased with low doses (1–3 Gy) but decreased at higher doses (5 Gy), suggesting a dose-dependent threshold effect. Similarly, shoot and root fresh weights displayed an initial increase followed by a decline with increasing doses. Biochemical parameters showed dose-dependent responses, with low to moderate doses (1–3 Gy) stimulating enzyme activities and high doses (5 Gy) inhibiting them. Gene expression analysis was focused on the following specific genes: thymol synthase, γ-terpinene synthase, and carvacrol synthase. Low to moderate doses increased the expression of these genes, resulting in increased production of bioactive compounds. However, higher doses had diminished effects or suppressed gene expression. Metabolite analysis demonstrated dose-dependent responses, with moderate doses enhancing secondary metabolite production, while higher doses provided limited benefits. These findings underscore the implications of using gamma radiation to enhance secondary metabolite production in plants and its potential applications in agriculture, medicine, and environmental science. The study emphasizes the potential of gamma radiation as an external stressor to influence plant responses and highlights the importance of understanding such effects in various fields.

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Vol.:(0123456789)
Environmental Science and Pollution Research
https://doi.org/10.1007/s11356-024-33269-y
RESEARCH ARTICLE
Exploring low‑dose gamma radiation effects onmonoterpene
biosynthesis inThymus vulgaris: insights intoplant defense
mechanisms
MojtabaKordrostami1· ForoughSanjarian2· SamiraShahbazi1· AliAkbarGhasemi‑Soloklui1
Received: 25 July 2023 / Accepted: 5 April 2024
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2024
Abstract
Thymus vulgaris, commonly known as thyme, is a plant renowned for producing monoterpenes. This study aimed to under-
stand the effects of low-dose gamma radiation, specifically in the range of 1–5Gy, on various traits of Thymus vulgaris,
providing context on its importance in agricultural and medicinal applications. The research explored morpho-physiological,
biochemical, and gene-expression responses in thyme plants under no gamma- and gamma-ray exposure conditions. The study
revealed complex relationships between gamma-ray doses and plant characteristics. In particular, shoot and root lengths ini-
tially increased with low doses (1–3Gy) but decreased at higher doses (5Gy), suggesting a dose-dependent threshold effect.
Similarly, shoot and root fresh weights displayed an initial increase followed by a decline with increasing doses. Biochemi-
cal parameters showed dose-dependent responses, with low to moderate doses (1–3Gy) stimulating enzyme activities and
high doses (5Gy) inhibiting them. Gene expression analysis was focused on the following specific genes: thymol synthase,
γ-terpinene synthase, and carvacrol synthase. Low to moderate doses increased the expression of these genes, resulting in
increased production of bioactive compounds. However, higher doses had diminished effects or suppressed gene expression.
Metabolite analysis demonstrated dose-dependent responses, with moderate doses enhancing secondary metabolite produc-
tion, while higher doses provided limited benefits. These findings underscore the implications of using gamma radiation to
enhance secondary metabolite production in plants and its potential applications in agriculture, medicine, and environmental
science. The study emphasizes the potential of gamma radiation as an external stressor to influence plant responses and
highlights the importance of understanding such effects in various fields.
Keywords Gamma radiation· Gene expression· Monoterpene biosynthesis· Plant physiology· Thymus vulgaris
Introduction
Gamma radiation, a form of electromagnetic radiation
encompassing X-rays, UV light, infrared light, microwaves,
and radio waves, possesses the most significant energy and
smallest wavelength among these forms of radiation (Shukla
and Mannheim 2020). With this characteristic, gamma rays
can penetrate matter at unparalleled depths compared to
other types of radiation (Chaudhary and Kumar 2023). The
primary source of gamma radiation lies with naturally occur-
ring and synthetic radioactive isotopes, which undergo radi-
oactive decay and emit gamma rays as a byproduct (Aladj-
adjiyan 2022). Cobalt-60 and radon-222 are two examples
of commonly encountered gamma sources employed across
various domains, including medicine and industry (Van Dyk
etal. 2020). Notably, gamma radiation exhibits ionizing
properties due to its sufficient energy to strip tightly bound
electrons from atoms, forming ions (Bakar etal. 2019).
As a result, prolonged exposure to gamma radiation risks
damaging living cells and tissues through mechanisms like
mutation and cell death (Bakar etal. 2019). It is essential to
differentiate between substantial and insignificant dosages
of gamma radiation concerning their potential consequences
on living organisms.
Responsible Editor: Gangrong Shi
* Forough Sanjarian
fsanjarian@nigeb.ac.ir
1 Nuclear Agriculture Research School, Nuclear Science
andTechnology Research Institute (NSTRI), Karaj, Iran
2 Plant Bioproducts Department, Institute ofAgricultural
Biotechnology, National Institute ofGenetic Engineering
andBiotechnology, Tehran, Iran

Environmental Science and Pollution Research
In contrast, high doses can lead to catastrophic outcomes,
and low-dose exposure remains a topic of continued research
(Dowlath etal. 2021). Apart from its practical applications,
such as medical imaging, cancer therapy, food sterilization,
and nuclear reactor operation, gamma radiation also plays
an integral role in environmental studies, focusing specifi-
cally on how it affects plant growth and development, with
particular attention paid to the monoterpene content pre-
sent in Thymus vulgaris (Venugopal 2011). Understanding
the implications of gamma radiation on plant life is crucial
for comprehending the aftermath of nuclear incidents and
designing future interstellar missions involving cultivated
crops exposed to cosmic gamma radiation (De Micco etal.
2022).
Plants are exposed to diverse forms of ionizing radiation
in their surroundings, including gamma radiation, which
originates from both natural (e.g., radon gas in soil) and
artificial sources (e.g., fallout from nuclear incidents or test-
ing) (Real etal. 2004). Investigating the consequences of
low-dose gamma radiation on plant health is crucial to evalu-
ating environmental risks and devising protective measures
for crops (Mothersill and Seymour 2022). The high-energy
photons emitted by gamma rays possess sufficient kinetic
energy to disrupt atomic structures within plants, forming
ions through ionization processes (Rasmidi etal. 2021).
These alterations can directly harm DNA, proteins, and other
cellular components. Additionally, ionizing radiation can
generate reactive oxygen species (ROS), leading to further
tissue damage (Duarte etal. 2023).
Nonetheless, plants exhibit redundant systems to mend
damaged cells and remove ROS, thereby minimizing adverse
effects. Furthermore, they adapt to radiation stress by modi-
fying their metabolic pathways and growth patterns (Ludov-
ici etal. 2020). At low doses, gamma radiation can elicit
varied responses in plants, ranging from promoting growth
enhancement (a phenomenon termed hormesis) to induc-
ing genetic changes, with some mutations potentially being
advantageous (Volkova etal. 2022; Vaiserman etal. 2021).
While the impact of low-dose gamma radiation on monoter-
penes in Thymus vulgaris remains relatively unexplored, we
aim to address this knowledge gap. Our investigation will
improve crop production efficiency and expand our com-
prehension of plant resistance in radioactive settings, which
could benefit both agriculture and space exploration initia-
tives (Hong etal. 2022). Long-term exposure to low-dose
radiation might display distinct characteristics compared to
brief exposure periods, so we expect our results to provide
valuable insights into these variations.
Thymus vulgaris, or thyme, is an evergreen, woody shrub
indigenous to the Mediterranean region. Belonging to the
Lamiaceae family, this plant shares a taxonomic affinity with
other herbs like mint, sage, and lavender (Vouillamoz and
Christ 2020). As a versatile crop, thyme has been employed
for culinary, medicinal, and decorative purposes throughout
history. Our investigation into the effect of gamma radiation
on Thymus vulgaris focuses mainly on its ability to synthe-
size monoterpenes, essential compounds responsible for its
distinctive fragrance and medicinal properties. By exam-
ining the molecular mechanisms underlying monoterpene
biosynthesis under gamma radiation exposure, we aim to
expand our comprehension of plant responses to environ-
mental stresses at a molecular level. These findings have
far-reaching implications for agricultural practices and fields
such as ecology and space exploration, where the resilience
of crops to adverse conditions plays a critical role (Coz-
zolino etal. 2023; Laftouhi etal. 2023).
Monoterpenes are a class of terpenes, naturally occurring
organic compounds produced by various plants, including
Thymus vulgaris. Monoterpenes consist of two isoprene
units and have a molecular formula of C10H16. They are
often volatile, meaning they readily evaporate at room tem-
perature and are a significant component of the essential oils
of many plants (Mabou and Yossa 2021). Thymus vulgaris
is a plant that relies heavily on monoterpenes for its sur-
vival. These organic compounds serve as its natural defense
against harmful predators and pathogens. They possess a
range of bioactive properties, such as repelling or poisoning
insects and other animals, and contain antimicrobial proper-
ties (Mahdavi etal. 2020). The volatile nature of monoter-
penes allows them to serve as signals attracting pollinators.
The unmistakable scent of plants like Thymus vulgaris
can be credited to their distinctive blend of monoterpenes.
Recent research suggests that these monoterpenes may assist
plants in adjusting to challenging surroundings, such as dry-
ness, hot temperatures, and radiation exposure (Yeshi etal.
2022). Research is ongoing, but it is believed that different
monoterpenes, such as those in Thymus vulgaris, may have
promising healing abilities. One important monoterpene in
thyme, thymol, has effectively fought against bacteria, fungi,
and inflammation.
Monoterpenes significantly enhance the savor and fra-
grance of herbs and spices, including thyme. This makes
them especially interested in the food and beverage indus-
try (Mohammadian Yasuj etal. 2022). Understanding how
environmental factors, such as gamma radiation, affect
monoterpene production in Thymus vulgaris can provide
valuable insights into plant adaptation and stress responses
and potential applications in agriculture, industry, and medi-
cine. The specific monoterpene produced depends on the
enzyme involved. For example, in Thymus vulgaris, thymol
is one of the primary monoterpenes produced. Following
their synthesis, monoterpenes can be stored within the plant
in various ways, often in specialized structures such as glan-
dular trichomes (Najar etal. 2021). They can be released in
response to various stimuli, such as herbivory, to fulfill their
roles in defense, signaling, and stress response.

Environmental Science and Pollution Research
There has been an increasing fascination with gamma
radiation, a formidable environmental agent known to elicit
significant physiological and genetic responses in plants
(Katiyar etal. 2022). Notably, the influence of low doses
of gamma radiation on plant secondary metabolism, par-
ticularly the biosynthesis of monoterpenes, remains largely
unexamined. Unraveling the effects of gamma radiation on
plant metabolism holds great significance as it can help
evaluate the implications of environmental radiation expo-
sure and reveal novel strategies for modulating secondary
metabolism for industrial or therapeutic applications. With
this goal in mind, we embarked upon a study investigating
the effect of low-dose gamma radiation on the expression of
monoterpene synthase genes and, subsequently, the forma-
tion of monoterpenes in plants.
Material andmethods
The Gene Bank Natural Resources, situated at the Research
Institute of Forests and Rangelands in Tehran, Iran, supplied
the thyme seeds. These seeds were subjected to γ-irradiation
doses of 1, 3, and 5Gy at the Nuclear Science and Technol-
ogy Research Institute in Karaj, Iran. To ensure the validity
of the results, each gamma radiation treatment was replicated
thrice. Discounting the control, 180 seeds were subjected to
γ radiation, each dosage involving three sets of 20 seeds.
The seeds underwent disinfection with 5% sodium hypochlo-
rite for 5min, followed by a rinse in sterilized water for an
equal amount of time. Their germination occurred on What-
man-grade 181 filter papers situated within Petri dishes.
The resulting seedlings were then relocated to pots filled
with sterilized peat moss and left there for 20days. This
method was chosen to replicate a controlled environment,
allowing for a clear assessment of the radiation’s effects.
All the plants were then grown in clay loam soil (consisting
of 21% sand, 20% silt, and 39% clay) within a greenhouse,
maintaining an average temperature of 20–25 °C, relative
humidity levels of 50–70%, a photoperiod of 16h, and a
photosynthetic photon flux density of 850–950µmol/m2/s.
After 4months, the pots were moved to a growth chamber
(courtesy of Weiss Umwelttechnik GMBH, Lindenstruth,
Germany). Here, they were kept under light for 6h (with a
photosynthetic photon flux density of 100–140µmol/m2/s)
at a temperature of 20–22 °C, followed by 8h of darkness
at 18–20°C and a relative humidity of 50–55%. Every 3 to
4days, the plants received irrigation through distilled water.
Following a month-long acclimatization period, young
leaves from five terminal nodes of each replicate were col-
lected from all the experimental groups four weeks after the
final treatment. These samples were immediately immersed
in liquid nitrogen for digestion and subsequently preserved
at a temperature of − 80°C for future experimentation about
terpene and RNA extractions.
Morphological evaluation
A month after the final treatment, specific characteristics
such as the length of the five terminal internodes, the length
and breadth of leaves, the ratio of leaf length to width, and
the length of new offshoots were determined using a ruler.
Additionally, the fresh weight (FW) and dry weight (DW)
of a sample of 100 leaves were gauged using a precise scale.
Total flavonoid andanthocyanin extraction
The overall flavonoid content was quantified through a
spectrophotometric procedure. A combination of aluminum
chloride (AlCl3) at 10% and a 1M potassium acetate solu-
tion was introduced to the methanol extract. The solution’s
absorbance was assessed at a 415 wavelength following
a half-hour incubation period at ambient temperature. A
standard calibration curve was created using quercetin as
a reference.
In the quantification of total anthocyanin, a 1% hydro-
chloric acid solution in methanol, referred to as acidic
methanol, was utilized for extraction. This process occurred
over a day at a cold temperature range of 3 to 5°C, with
periodic agitation. The extracted solution was centrifuged
at 12,000 rotations per minute for 20min. The absorbance
of the resulting clarified extract was assessed against control
at 530nm (the absorption peak for anthocyanin) and 657nm
(the absorption peak for chlorophyll degradation products in
acidic methanol). The anthocyanin content of each extract
was determined based on the absorbance calculated through
the equation: A = A530 − (0.25 × A657), where A530 and
A657 represent the solution’s absorbance at 530nm and
657nm wavelengths, respectively.
RNA extraction andqPCR analyses
Using a TissueRuptor (manufactured by Qiagen, Ger-
many), leaves were ground into a homogenous mixture in
liquid nitrogen. Polyvinylpolypyrrolidone (obtained from
Sigma-Aldrich, Steinheim, Germany) was introduced to
bind polysaccharides and polyphenols. The choice to uti-
lize the GeneJET Plant RNA Purification Mini Kit (pro-
duced by Thermo Fisher Scientific Inc.) for RNA extrac-
tion was due to its efficiency and reliability in yielding
high-quality RNA. The purity and concentration of the
RNA were assessed by measuring the absorbance of the
samples at 260, 280, and 230nm, utilizing a spectropho-
tometer (produced by Eppendorf, Hamburg, Germany).
Specific genes for RNA extraction and qPCR analyses,
such as γ-terpinene synthase (TPS), thymol synthase

Environmental Science and Pollution Research
(CYP71D178, CYP71D179, and CYP71D182), and carvac-
rol synthase (CYP71D180 and CYP71D181), were selected
based on their known roles in monoterpene biosynthesis in
Thymus vulgaris. The RNA concentration ranged between
800 and 1200ng/µl. However, all samples were diluted
with DEPC-treated water to reach a uniform concentra-
tion of 800ng/µl. RNase-free DNAse I (also from Thermo
Fisher Scientific Inc.) was used to remove any remaining
DNA in the samples. Synthesis of the first strand cDNA
was accomplished using oligo (dT)18 primer and a reverse
transcriptase enzyme (provided by Thermo Fisher Scien-
tific Inc.) (Rabiei etal. 2018). A negative control (with-
out reverse transcriptase) was included using total RNA
to check for any contamination from genomic DNA. A
polymerase chain reaction (PCR) was implemented to con-
firm the sizes of the amplified products from each of the
monoterpene synthase primers and detect the presence of
DNA contamination. This procedure was completed in a
50-µl final volume that contained 2µl cDNA or a non-RT
control, 5µl of 10 × PCR buffer, 1µl of a 10-mM mix
of dNTPs, 1.5µl of each specific gene primer, 3µl of
25-mM MgCl2, 0.5µl of Taq DNA polymerase (5 U/µl),
and nuclease-free water. The PCR process was executed
utilizing a Mastercycler gradient (a product of Eppendorf,
Hamburg, Germany) and followed the following sequence:
initial heating at 95°C for 5min, then 35 cycles at 94°C
for 30s, 60°C for 1min, and 70°C for 1min, with a final
extension at 72°C for 7min.
The amplified products exhibited distinct bands on
1.5% agarose gels, consistent with the anticipated frag-
ments, with no signs of unexpected products. The PCR
products had lengths of 90bp for CYP71D181, 99bp for
CYP71D178, 108bp for CYP71D179/182, 114bp for
CYP71D180, and 121bp for the ovEFalpha genes. The
relative transcript levels of the monoterpene synthase
genes, which include γ-terpinene synthase (TPS), thymol
synthase (CYP71D178, CYP71D179, and CYP71D182),
and carvacrol synthase (CYP71D180 and CYP71D181),
were calculated using the Normalized Expression Method
(fold change) as outlined by Pfaffl (2001). The Origanum
vulgare L. eukaryotic transcription elongation factor 1
alpha cDNA (ovEF1alpha, accession number, GU385981)
was used as a constant internal control. Real-time PCR
was conducted using an iCycler (provided by Bio-Rad,
Hemel Hempstead, U.K.) and SYBR green I (from Thermo
Fisher Scientific Inc.). Each 25µl RT-PCR reaction con-
sisted of cDNA, equating to 1.6ng of the total RNA. PCR
thermal cycling was carried out as follows: an initial heat
at 95°C for 10min, then 40 cycles at 95°C for 30s, at
60°C for 1min, and at 70°C for 1min. The melting tem-
perature ranged from 55 to 95°C. The primers used for
PCR and qRT-PCR reactions were gene-specific (Rabiei
etal. 2018).
Gas chromatography analysis ofplant volatiles
To create a fine powder, plant material that had been fro-
zen was crushed using a mortar and pestle. This powdered
material (100mg) was then submerged in an ethyl acetate
pentane (2:1 ratio) solution and left to soak for 24h at ambi-
ent temperature. The answer was clarified using cellulose
wadding placed on a Pasteur pipette. Volatile compounds
were subjected to chromatographic analysis with a gas chro-
matograph (Agilent Hewlett-Packard 6890, Agilent/J and W
Scientific, Folsom, CA, USA) equipped with a flame ioniza-
tion detector (FID) and a splitless injector. This process was
conducted at the Research Institute of Forests and Range-
lands in Tehran. The compounds were separated using a DB-
WAX capillary column (0.25mm ID × 30m, Agilent/J, and
W Scientific, Folsom, CA, USA), which had a film thickness
of 250mm. The temperature protocol used was as follows:
initial temperature of 80°C for 2min, then increased by
10°C/min to 140°C for 1min, subsequently, increased by
4°C/min to 190°C for 2min, and finally, increased by 2°C/
min to 210°C for 2min. Helium was utilized as the carrier
gas with a 1.0-mL/min flow rate. The detector gasses com-
prised hydrogen and air, with a flow rate of 40mL.min−1.
The detector and injector temperatures were set to 210 and
200°C, respectively. The correlation between terpene con-
tent and transcript levels was determined using Spearman’s
rank correlation coefficient.
Statistical analysis
The experimental setup was structured as a completely ran-
domized design, including three replications. To ascertain
the statistical significance of the differences between average
values, a one-way analysis of variance (ANOVA) paired with
LSD (least significant difference) Tukey tests was employed.
The choice of LSD Tukey tests was justified by their ability
to control the Type-I error rate while comparing multiple
group means. Any significant disparities were assessed at a
significance level of 1%.
Results
Effect oflow dose gamma radiation
onmorpho‑physiological andbiochemical traits
The study revealed threshold effects on shoot and root
lengths at specific gamma-ray doses. (Fig.1a). In the con-
trol group (C) with no gamma-ray exposure, the average
shoot length was measured at 11.33cm. Upon exposure to a
gamma-ray dose of 1Gy, a slight increase in average shoot
length was observed, with the length reaching 11.83cm,
indicating an approximate 4% increase compared to the

Environmental Science and Pollution Research
control group. When the gamma-ray dose was increased
to 3Gy, a significant rise in the average shoot length was
observed, reaching 17.33cm. This denotes a substantial
increase of approximately 53% compared to the control
group. However, at a gamma-ray dose of 5Gy, the aver-
age shoot length returned to 11.83cm, which is the same
as the shoot length observed at a gamma-ray dose of 1Gy
and represents a significant decrease from the shoot length
observed at 3Gy. These findings suggest a complex relation-
ship between gamma-ray dose and shoot length, with an ini-
tial increase followed by a plateau and a significant decrease.
These results suggest a potential threshold effect, where
shoot length increases initially but declines after exceeding
a certain gamma-ray exposure level. For the root length,
in the control group (C), the measurement was recorded as
12.00cm (Fig.1b). With exposure to a gamma-ray dose
of 1Gy, there was a considerable increase in root length,
which was recorded at 17.67cm. This signifies an increase
of approximately 47% compared to the control group. How-
ever, a slight decrease in root length was observed when
the gamma-ray dose was increased to 3Gy, with the length
reducing to 15.77cm. Despite this decrease, the root length
at 3Gy was still approximately 31% higher than the con-
trol group. At a gamma-ray dose of 5Gy, a further decline
in root length was observed, with the length measured at
13.00cm. This length represents an increase of approxi-
mately 8% compared to the control group, but a decrease
of 26 and 18% compared to the root lengths is observed
at gamma-ray doses of 1Gy and 3Gy, respectively. These
results suggest a complex relationship between gamma-ray
dose and root length, with an initial increase followed by a
decrease as the dose of gamma rays increases. This change
indicates a similar threshold effect in root growth, where
it begins to decline beyond a certain level of gamma-ray
exposure. Exploring potential reasons for these changes,
the initial increases at lower doses might be attributed to
radiation-induced stimulation, while the decreases at higher
doses suggest radiation damage.
The study results showed that in the control group (C),
the shoot fresh weight was measured at 0.55g (Fig.1c).
When exposed to a gamma ray dose of 1Gy, there was a
noticeable increase in shoot fresh weight to 0.69g, marking
Fig. 1 Impact of low-dose
gamma radiation on shoot and
root lengths, and fresh weight in
thyme plants

Environmental Science and Pollution Research
an approximate rise of 25% compared to the control group.
Further exposure to a gamma ray dose of 3Gy led to a sub-
stantial increase in shoot fresh weight, which was recorded
at 1.06g. This indicates a remarkable increase of approxi-
mately 93% compared to the control group. However, at a
gamma ray dose of 5Gy, the shoot fresh weight decreased
to 0.84g. Despite this decrease, the weight at 5Gy was still
approximately 53% higher than the control group, but it was
about 21% lower than the weight observed at a gamma ray
dose of 3Gy. These findings suggest a complex relationship
between gamma-ray dose and shoot fresh weight, with an
initial increase followed by a decrease as the dose of gamma
rays increases. This could suggest a threshold effect where
shoot fresh weight starts to decline beyond a certain level of
gamma-ray exposure. This trend indicates a threshold effect
in shoot fresh weight as well.
The results assessed the impact of different gamma-ray
doses on root fresh weight (Fig.1d). In the control group
(C) with no gamma-ray exposure, the root fresh weight was
measured at 0.19g. Upon exposure to a gamma ray dose of
1Gy, there was a significant increase in root fresh weight,
with the weight recorded at 0.43g. This indicates an approx-
imate increase of 126% compared to the control group. How-
ever, when the gamma-ray dose was increased to 3Gy, a
noticeable decrease in root fresh weight was observed, with
the weight measured at 0.17g. Despite this decrease, the
weight at 3Gy was still approximately 11% lower than the
control group. At a gamma-ray dose of 5Gy, the root fresh
weight slightly increased to 0.21g. Despite this increase, the
weight at 5Gy was still approximately 11% higher than that
observed at a gamma ray dose of 3Gy but about 51% lower
than that observed at a gamma ray dose of 1Gy. This also
suggests a threshold effect where root fresh weight starts to
decline beyond a certain level of gamma-ray exposure.
The effect of gamma radiation doses on the number of
nodes per plant was evaluated, and the outcomes are sum-
marized in Fig.2a. The control group (C) had an average of
13 nodes per plant. Interestingly, the number of nodes per
plant increased with higher doses of gamma radiation. When
exposed to a dose of 1Gy, the number of nodes per plant
remained the same as the control, at 13 nodes. However, a
notable increase was observed with higher radiation doses.
A dose of 3Gy led to an average of 15 nodes per plant,
indicating an increase of approximately 15.4% compared to
the control group.
Further, plants exposed to a dose of 5Gy had an average
of 16 nodes per plant, an increase of approximately 23.1%
compared to the control group. These results suggest that
gamma radiation can potentially influence plant nodal devel-
opment at higher doses. This study examined the impact
of varying gamma-ray doses on the number of secondary
branches per plant. The effect of varying doses of gamma
radiation on the number of secondary branches in the study
plants was observed (Fig.2b). Under control conditions
(Ctlr), where no gamma radiation was applied, the number
of secondary branches was found to be 2. The introduction of
gamma radiation showed a noticeable increase in the num-
ber of secondary branches. At a dose of 1Gy, the number
of secondary branches increased to 3. The most significant
increase was observed at a dose of 3Gy, with the number of
secondary branches rising dramatically to 13. However, as
the radiation dose was increased further to 5Gy, a reduction
was noted in the number of secondary branches, decreas-
ing to 8. This suggests a possible threshold effect at doses
beyond 3Gy, where the higher radiation dose does not result
in a proportional increase in secondary branching but may
instead induce inhibitory or damaging effects.
This research analyzed the impact of different levels of
gamma-ray dosage on CO2 absorption (Fig.3a). A complex
relationship between gamma-ray dosage and CO2 absorp-
tion was observed, with an initial decrease, followed by a
slight increase and a significant decline, indicating a thresh-
old effect in CO2 absorption capacity. The CO2 absorption
level when there was no gamma-ray dose was recorded at
576.33ppm. An initial gamma-ray dose of 1Gy decreased
CO2 absorption to 553.67ppm, indicating an initial decrease
of approximately 4%. This was followed by a slight increase
in absorption levels to 577.33ppm at a gamma-ray dose of
3Gy, effectively surpassing the absorption level of the con-
trol group. However, a dramatic drop in CO2 absorption was
observed at a gamma-ray dose of 5Gy. The absorption level
recorded was 524.90ppm, approximately 9% lower than the
control level. This suggests a possible threshold effect where
the CO2 absorption capacity starts to decrease significantly
beyond a certain level of gamma-ray dose. These findings
suggest a complex relationship between gamma-ray dosage
and CO2 absorption, with an initial decrease followed by
Fig. 2 Impact of low-dose gamma radiation on the number of nodes
and number of secondary branches in thyme plants

Environmental Science and Pollution Research
a slight increase and then a significant decline. The study
also evaluated the effect of varying gamma-ray dosage lev-
els on outside CO2 (Fig.3b). The baseline outside CO2 (C,
the control group) with no gamma-ray exposure was meas-
ured at 540.67ppm. Upon exposure to a gamma-ray dose of
1Gy, there was an increase in outside CO2 to 557.33ppm,
representing an approximate increase of 3%. As the gamma-
ray dose was increased to 3Gy, the outside CO2 further
increased to 574.67ppm, marking a steady trend of rising
outside CO2 with increasing gamma-ray dose up to this
point. However, at a gamma-ray dose of 5Gy, a notable
decrease in outside CO2 was observed, with the level drop-
ping to 521.67ppm. This represents a decrease of approxi-
mately 4% from the control group’s outside CO2 level. The
results suggest a dynamic relationship between gamma-ray
dose and outside CO2, with an initial increase followed by a
subsequent decrease. The data indicates a potential threshold
effect where the outside CO2 decreases beyond a certain
level of gamma-ray exposure.
The study’s results examined the influence of differ-
ent gamma-ray doses on the net photosynthesis rate (Pn)
(Fig.3c). In the control group (C) with no gamma-ray
exposure, the net photosynthesis rate was measured at
16.20µmol.m−2.s−1. Upon exposure to a gamma-ray dose
of 1Gy, there was a considerable increase in the net photo-
synthesis rate to 24.30µmol.m−2.s−1, signifying a roughly
50% increase compared to the control group. Further, an
increase in the gamma-ray dose to 3Gy resulted in a sig-
nificant rise in the net photosynthesis rate to 37.33µmol.
m−2.s−1, more than double the rate observed in the control
group. Interestingly, at a gamma-ray dose of 5Gy, the net
photosynthesis rate increased even more, reaching a level of
39.37µmol.m−2.s−1. This represents an approximate 143%
increase compared to the control group. The results indi-
cate a positive correlation between gamma-ray dose and the
net photosynthesis rate. The data suggest that increasing
gamma-ray doses might enhance the photosynthetic activ-
ity up to a certain point, as evidenced by the increased net
photosynthesis rate at a dose of 5Gy. The study’s results
evaluated the impact of varying gamma-ray doses on photo-
synthetically active radiation (PAR) (Fig.3d). In the control
group (C), the PAR was measured at 322.67µmol.m−2.s−1.
Fig. 3 Impact of low-dose
gamma radiation on photo-
synthetic parameters in thyme
plants

Environmental Science and Pollution Research
When exposed to a gamma-ray dose of 1Gy, there was a
substantial increase in PAR to 469.33µmol.m−2.s−1, mark-
ing an approximately 45% increase compared to the control
group. A further increase in the gamma-ray dose to 3Gy
resulted in a slight rise in PAR to 472.67µmol.m−2.s−1,
maintaining a high level of photosynthetically active radia-
tion. However, a significant decrease in PAR was observed
at a gamma-ray dose of 5Gy, plummeting to 249.49µmol.
m−2.s−1. This represents a decrease of approximately 23%
from the control group’s PAR level. These results suggest
a complex relationship between gamma-ray dose and PAR.
While there is an initial increase in PAR with low to moder-
ate gamma-ray doses, a significant decline is observed at
higher doses. This data indicates a possible threshold effect,
where the PAR starts to drop dramatically beyond a certain
level of gamma-ray exposure.
The influence of varying gamma radiation doses on
anthocyanin content in plants was examined, with the find-
ings detailed in Fig.4a. In the control group (C), the antho-
cyanin content was measured at 0.06mg.g−1 FW. Interest-
ingly, the anthocyanin content exhibited a varying pattern
in response to different doses of gamma radiation. Plants
exposed to a gamma radiation dose of 1Gy showed a slight
decrease in anthocyanin content to 0.05mg.g−1 FW, rep-
resenting an approximate decrease of 16.7% compared to
the control group. However, with a gamma radiation dose
of 3Gy, the anthocyanin content rose to 0.07mg.g−1 FW,
about 16.7% higher than the control group. This suggests
that intermediate doses might positively influence anthocya-
nin production in plants.
On the contrary, a significant reduction in anthocyanin
content was observed when the gamma radiation dose was
increased to 5Gy. The anthocyanin content dropped to
0.03mg.g−1 FW, a decrease of approximately 50% com-
pared to the control group. These results indicate a com-
plex relationship between gamma radiation dose and plant
anthocyanin production. This study analyzed the impact of
various gamma-ray doses on flavonoid content (Fig.4b). The
control group (C) that received no gamma-ray exposure had
a flavonoid content of 0.13mg QE.g−1. When exposed to a
gamma-ray dose of 1Gy, there was a marginal increase in
flavonoid content, measured at 0.14mg QE.g−1, indicating
a rise of approximately 7.7% compared to the control group.
However, with gamma-ray doses of 3 and 5Gy, a decrease
Fig. 4 Impact of low-dose
gamma radiation on anthocya-
nins, flavonoids, and antioxidant
enzymes in thyme plants

Environmental Science and Pollution Research
in flavonoid content was observed, with both doses resulting
in a flavonoid content of 0.12mg QE.g−1. This represents
a decrease of approximately 7.7% compared to the control
group and approximately 14.3% compared to the flavonoid
content at 1Gy. This data suggests that the flavonoid content
in plants might be influenced by the dose of gamma rays,
with an initial increase followed by a decrease as the dose
of gamma rays increases.
This study evaluated the effects of varying gamma-ray
doses on polyphenol oxidase (PPO) activity (Fig.4c). In
the control group (C), the PPO activity was recorded at 8.34
U.mg−1. When exposed to a gamma-ray dose of 1Gy, there
was a slight increase in PPO activity, reaching 8.81 U.mg−1,
an increase of approximately 5.6% compared to the con-
trol group. Further, upon increasing the gamma-ray dose to
3Gy, a significant increase in PPO activity was observed,
with the activity reaching 9.93 U.mg−1, marking a significant
increase of approximately 19.1% compared to the control
group. However, when the gamma-ray dose was increased
to 5Gy, the PPO activity decreased slightly to 8.86 U.mg
−1, which, while higher than the control group by approxi-
mately 6.2%, was lower than the activity recorded at 3Gy by
approximately 10.8%. These results suggest a complex rela-
tionship between gamma-ray dose and PPO activity, with an
initial increase followed by a slight decrease at higher doses.
This study assessed the impact of different gamma-ray doses
on peroxidase (POD) activity (Fig.4d). The control group
(C) was not exposed to gamma rays and had a baseline POD
activity of 27.41 U.mg−1. Upon exposure to a gamma-ray
dose of 1Gy, there was a considerable decrease in POD
activity to 20.04 U.mg−1. This represents a decrease of
approximately 27% compared to the control group. However,
a significant increase in POD activity was observed when the
gamma-ray dose was increased to 3Gy. The activity rose to
32.39 U.mg−1, marking an increase of approximately 18%
compared to the control group and a substantial increase
of 62% compared to the activity observed at 1Gy. When
the gamma-ray dose was further increased to 5Gy, POD
activity decreased to 23.57 U.mg−1. Despite this decrease,
the POD activity at 5Gy was still approximately 16% lower
than the control group and about 17% higher than the activ-
ity at 1Gy. These results illustrate a complex relationship
between gamma-ray dose and POD activity, with an initial
decrease, followed by an increase, and then a decrease as the
gamma-ray dose increases.
Effect oflow‑dose gamma radiation onthegene
expression patterns
The influence of gamma radiation on thymol synthase
(CYP71D178, CYP71D179, and CYP71D182) gene expres-
sion linked to thymol production was evaluated. Figure5
details the change in expression following different radiation
doses. A dose-dependent relationship between gamma radia-
tion and these gene expressions was observed, emphasizing
Fig. 5 Impact of low-dose
gamma radiation on gene
expression patterns in thyme
plants

Environmental Science and Pollution Research
the nuanced effect of gamma radiation on gene expression
and potential applications in plant biotechnology.
At 1Gy, expression increased slightly (0.956 units); at
3Gy, a substantial upregulation (3.374 units) was observed.
However, at 5Gy, expression declined (2.139 units), though
it remained higher than the control group. These findings
suggest a dose-dependent relationship between gamma
radiation and thymol synthase expression. Additionally,
γ-terpinene synthase (TPS) gene expression, responsible for
γ-terpinene production, was examined. At 1Gy, expression
modestly increased (1.131 units). At 3Gy, a significant rise
(4.237 units) was noted, while at 5Gy, a dramatic increase
(10.544 units) was seen, emphasizing gamma radiation’s
dose-dependent impact on γ-terpinene synthase expression.
Finally, carvacrol synthase activity, linked to carvacrol pro-
duction, was investigated. At 1Gy, activity was 0.403 units.
A significant increase to 2.635 units was observed at 3Gy,
but activity reduced to 1.32 units at 5Gy. This suggests that
moderate gamma radiation boosts carvacrol synthase activ-
ity, but higher doses might have an inhibitory effect. Overall,
these results highlight the potential of gamma radiation in
modulating the production of bioactive compounds in plants,
although the effect appears to be dose-dependent.
Effect oflow‑dose gamma radiation
onthesecondary metabolites
The results demonstrate the impact of different gamma-ray
doses on the thymol content in the experimental samples
(Fig.6a). The control group (C) had a thymol content of
150ppm. Upon exposure to a gamma-ray dose of 1Gy, the
thymol content slightly increased to 160ppm. However, a
notable enhancement in thymol content was observed at a
gamma-ray dose of 3Gy, reaching 230ppm. Surprisingly,
the thymol content at a higher dose of 5Gy returned to
160ppm, similar to the 1Gy dose. These findings indicate
that gamma-ray exposure can influence the thymol content,
but the response may not follow a linear pattern. While
a moderate gamma-ray dose of 3Gy promoted a signifi-
cant increase in thymol content, the control group and the
higher dose of 5Gy exhibited comparable thymol levels.
The results illustrate the impact of different gamma-ray
doses on the carvacrol content in the experimental sam-
ples (Fig.6b). The control group (C) exhibited a carvac-
rol content of 14ppm. Upon exposure to a gamma-ray
dose of 1Gy, the carvacrol content slightly increased to
16ppm. However, a significant enhancement in carvacrol
content was observed at a gamma-ray dose of 3Gy, reach-
ing 19ppm.
Interestingly, the carvacrol content decreased to
17ppm at a higher dose of 5Gy. These findings suggest
that gamma-ray exposure can influence carvacrol content,
albeit the response may not follow a linear trend. While
a moderate gamma-ray dose of 3Gy led to a substantial
increase in carvacrol content, both the control group and
the higher dose of 5Gy exhibited comparable carvacrol
levels.
In the control group (C), the γ-terpinene content was
measured at 75ppm (Fig.6c); upon exposure to a gamma-
ray dose of 1Gy, the γ-terpinene content increased to
113ppm, indicating a noticeable induction of γ-terpinene
production. A significant enhancement in γ-terpinene con-
tent was observed at a gamma-ray dose of 3Gy, reaching
424ppm. This finding suggests that a moderate dose of
gamma rays can significantly enhance the production of
γ-terpinene. However, interestingly, the γ-terpinene con-
tent decreased to 100.5ppm at a higher dose of 5Gy. This
suggests that there might be a dose-dependent response
to gamma radiation, where excessive exposure to gamma
rays could hurt γ-terpinene production. These results high-
light the potential of gamma-ray treatments to influence
the γ-terpinene content in the studied system. These results
highlight the potential of gamma radiation in modulating
the production of bioactive compounds in plants.
Fig. 6 Impact of low-dose
gamma radiation on secondary
metabolites in thyme plants

Environmental Science and Pollution Research
The correlation andcluster analysis
The correlation analysis was performed to examine the
relationships between the studied traits. The correlation
coefficients between the traits are presented in Fig.7.
The analysis revealed several significant correlations
among the traits. The total fresh weight (TFW) showed a
strong positive correlation with shoot fresh weight (SFW)
(r = 0.88) and a moderate positive correlation with root
length (RL) (r = 0.60). Additionally, a weak positive cor-
relation was observed between TFW and the number of
nodes per plant (NN) (r = 0.36). The root fresh weight
(RFW) exhibited a weak positive correlation with TFW
(r = 0.27) and a weak positive correlation with shoot
fresh weight (SFW) (r = 0.42). Shoot fresh weight (SFW)
displayed a strong positive correlation with total fresh
weight (TFW) (r = 0.88) and a moderate positive corre-
lation with root length (RL) (r = 0.73). Moreover, SFW
showed a weak positive correlation with the number of
nodes per plant (NN) (r = 0.42). The root length (RL)
exhibited a moderate positive correlation with shoot fresh
weight (SFW) (r = 0.73) and a weak positive correlation
with total fresh weight (TFW) (r = 0.60). The number of
nodes per plant (NN) showed a weak positive correla-
tion with total fresh weight (TFW) (r = 0.36), shoot fresh
weight (SFW) (r = 0.42), and root length (RL) (r = 0.62).
The number of secondary branches (NSB) displayed
weak positive correlations with root fresh weight (RFW)
(r = 0.32) and shoot fresh weight (SFW) (r = 0.27). The
shoot length (PH) exhibited weak positive correlations
with total fresh weight (TFW) (r = 0.29), shoot fresh
weight (SFW) (r = 0.34), and the number of nodes per
plant (NN) (r = 0.68).
Cluster analysis was employed to classify the treat-
ments under study (Fig.8). The analysis yielded three
distinct clusters. The first cluster comprised the control
(C) and 1Gy treatment. This co-clustering emerged due
to the close resemblance these two treatments bore. Upon
inspection, it was observed that most of the traits exhib-
ited by these treatments were identical, indicating similar
impacts on the studied subjects. The second cluster was
unique because it consisted solely of the 5-Gy treatment.
This treatment distinguished itself by recording the lowest
values across all traits. This implies that the 5-Gy treat-
ment was the least effective among the treatments under
consideration, given its consistently low performance.
Finally, the third cluster, similar to the second, was a sin-
gleton cluster consisting of only the 3Gy gamma radiation
treatment.
Contrary to the 5-Gy treatment, this treatment achieved
the highest values for almost all traits. This indicates a
superior performance of the 3-Gy gamma radiation treat-
ment to the other treatments. The results suggest its higher
efficacy in inducing desirable outcomes across most stud-
ied traits. The clustering of treatments thus provides valu-
able insights into their relative performance based on the
range of studied traits. This analysis revealed significant
correlations among traits and distinct clusters based on
treatment effectiveness, providing insights into their rela-
tive performance and the underlying mechanisms.
Fig. 7 Correlation heatmap of
studied traits in thyme plants

Environmental Science and Pollution Research
Discussion
The results of this study demonstrate a fascinating interplay
between gamma-ray doses and plant growth, as reflected in
shoot length and fresh weight. The observed increase in shoot
length and fresh weight at lower doses of gamma radiation (1
and 3Gy) indicates that gamma radiation might stimulate plant
growth up to a certain point. This stimulation may be attributed
to radiation-induced hormesis, where low-dose radiation trig-
gers positive biological responses. Such hormetic responses
could involve the upregulation of stress-related genes and the
activation of protective cellular pathways, leading to enhanced
growth and stress tolerance (Calabrese 2002). The substantial
increase in shoot length and fresh weight observed at a gamma-
ray dose of 3Gy suggests a strong hormetic response, where a
low-stress level stimulates a beneficial growth response (Beyaz
etal. 2016; McBride and Schaue 2020). This growth enhance-
ment is likely mediated through the activation of growth-pro-
moting hormones, such as gibberellins and cytokinins, and
stress-response pathways that lead to cell proliferation and
elongation (Dwimahyani and Widiarsih 2010).
Additionally, low-dose gamma radiation may induce
changes in the plant’s antioxidant defense system, enhanc-
ing its ability to cope with oxidative stress (Kovacs and
Keresztes 2002). Gamma radiation, even in low doses, can
cause ionization, disrupting the regular atomic structure
in cells (Ali etal. 2015). However, when the exposure is
at a lower dosage, plants can activate defense and repair
mechanisms, which in some cases might stimulate growth
instead of causing damage (Iglesias-Andreu etal. 2012).
The effect on shoot length specifically can be linked to
cellular division and differentiation in the meristematic tis-
sues of the plant. In normal growth, cells in the apical mer-
istem divide and differentiate, adding to the shoot’s length
(Dwimahyani and Widiarsih 2010). Exposure to low-dose
gamma radiation could potentially stimulate the rate of cell
division or alter the patterns of differentiation, increasing
shoot length. This might be due to the radiation-induced
expression of genes involved in cell cycle regulation and
differentiation (Wang etal. 2020). This increase might also
result from changes in plant hormones, as the balance of
hormones like auxins, cytokinins, and gibberellins plays
a crucial role in determining plant size and shape (Majeed
etal. 2018). Gamma radiation can change these hormones’
synthesis or signaling pathways, causing variations in
shoot growth. However, the return of shoot length to the
levels observed at 1Gy upon exposure to a gamma-ray
dose of 5Gy suggests a threshold effect. This implies that
the plant’s adaptive mechanisms might be overwhelmed
beyond a certain level of radiation stress, leading to a halt
in the enhanced growth response. Such a threshold effect
could result from excessive DNA damage, disruption of
cellular homeostasis, or overwhelming the plant’s antioxi-
dant defense system, leading to a reduction in growth (Gill
etal. 2015). The radiation might cause damage to the cel-
lular structures or disrupt the hormonal balance, leading to
a decrease in shoot elongation (Potters etal. 2007). These
findings highlight the dual nature of radiation as both a
stimulator and inhibitor of plant growth, depending on the
dosage. Understanding this delicate balance is crucial, as it
has implications for agricultural practices, particularly in
regions affected by radiation exposure (Calabrese 2002).
Fig. 8 Cluster heatmap of stud-
ied treatments in thyme plants

Environmental Science and Pollution Research
The observed results highlight an intricate interplay
between gamma-ray doses’ root length and fresh weight,
demonstrating a pattern that is not strictly linear. A marked
increase in root length and fresh weight after exposure to a
gamma-ray dose of 1Gy suggests that low-level radiation
could stimulate root growth or enhance nutrient absorption,
resulting in increased weight. This phenomenon might be
explained by the activation of specific genes responsible for
root development and nutrient transporters, which in turn
could increase the efficiency of nutrient uptake and assimi-
lation (Singh etal. 2013). This aligns with the concept of
radiation hormesis, which suggests that low doses of radia-
tion can have beneficial effects (Calabrese 2002; Volkova
etal. 2022). However, the subsequent decrease in root length
and fresh weight at 3Gy, followed by a slight recovery at
5Gy, adds complexity to this pattern. The decrease might
be due to the high-energy gamma rays causing cell damage
or altering metabolic processes, which could impede growth
and reduce root length and fresh weight (Kovacs and Keresz-
tes 2002; Ali etal. 2015). At higher doses, radiation may
disrupt the integrity of the root cell membranes, leading to
cellular dysfunction and reduced growth (Jolly and Meyer
2009). This decline suggests that beyond a certain radiation
threshold, the harmful effects of radiation exposure poten-
tially outweigh the beneficial hormetic effects, leading to
reduced growth and lower fresh weight. The slight increase
in root fresh weight at 5Gy compared to 3Gy could be due
to various factors, such as activation of repair mechanisms
or adaptive responses in the plant to cope with higher radia-
tion stress. At this higher dose, the plants may have acti-
vated secondary defense mechanisms, such as the produc-
tion of protective antioxidants, which could partly mitigate
the damaging effects of radiation (Ulukapi and Nasircilar
2022). However, the weight at 5Gy was still significantly
lower than that at 1Gy, indicating that the overall trend is
decreased in root fresh weight with increasing the gamma-
ray dose beyond the initial hormetic range. These findings
underscore the potential sensitivity of root growth and devel-
opment to gamma-ray doses and the possible existence of
a threshold effect, where root fresh weight starts to decline
beyond a certain level of radiation exposure. Understand-
ing these dose-dependent effects is crucial for leveraging
gamma radiation in agricultural practices and plant stress
physiology research.
Our findings reveal an intriguing response of plants to
gamma radiation exposure, particularly in nodal develop-
ment and the number of secondary branches per plant. It
appears that a threshold of gamma radiation, in this case
beyond 1Gy, is necessary to stimulate an increase in the
number of nodes per plant. This response could be linked
to the radiation-induced activation of meristematic activity
in the nodes, leading to increased cell division and growth
(Jones etal. 2020). The consistency of the increase at both
3 and 5Gy doses suggests that this is not a random occur-
rence but rather a systematic response to the radiation (Fait
etal. 2020). This increased nodal development could be a
plant stress response (Zhang etal. 2020). Gamma radiation,
as a form of ionizing radiation, can cause damage to cells
and DNA (Mavragani etal. 2019). In response, plants may
accelerate their growth and development to offset potential
damage, leading to increased nodes (Burroughs etal. 2023).
This phenomenon, known as stress-induced morphogenesis,
is observed in various plant species in response to differ-
ent stressors (Kouhen etal. 2023). The radiation-induced
expression of genes related to cell cycle regulation and
growth hormones such as cytokinins could be contributing
factors (Park and Runkle 2017). However, it is worth noting
that the increase in nodes at higher radiation doses might
indicate a stimulated growth response. Still, it does not nec-
essarily translate into overall plant health or productivity.
Other factors, such as the overall growth rate, plant bio-
mass, and reproductive success, need to be considered when
assessing the impact of gamma radiation on plant health
and productivity (Bado etal. 2023). Furthermore, while this
study suggests that higher doses of gamma radiation can
stimulate nodal development and the number of secondary
branches per plant, it is crucial to consider potential long-
term effects. Prolonged exposure to high levels of gamma
radiation could lead to harmful mutations and adverse effects
on overall plant health. These dose-dependent effects and the
balance between beneficial and harmful responses under-
score the complexity of plant responses to radiation stress,
offering valuable insights for future research in plant biology
and crop improvement strategies (Katiyar etal. 2022).
The study findings highlight the complex relationship
between gamma-ray dosage and CO2 absorption. Initial
exposure to gamma rays (1Gy) led to a decrease in CO2
absorption, suggesting that low levels of gamma rays may
harm the ability of the system to absorb CO2 efficiently. This
decline could be linked to radiation-induced alterations in
photosynthetic efficiency or damage to the stomatal appa-
ratus, which is crucial for CO2 uptake (Choi etal. 2021).
Interestingly, an increase in the gamma-ray dose to 3Gy
resulted in a slight elevation in CO2 absorption compared
to the control group. This observation could indicate a hor-
metic response, where low-to-moderate stress levels, such
as radiation, may stimulate a protective response and pro-
mote adaptability in the system. At this dose, gamma radia-
tion may trigger a compensatory increase in photosynthetic
activity or upregulate CO2 fixation pathways (Jaiswal etal.
2021). However, the significant decrease in CO2 absorption
at a gamma-ray dose of 5Gy suggests that there might be
a threshold beyond which the system can no longer cope
with the radiation-induced stress, leading to a decline in CO2
absorption capacity. This could be due to extensive dam-
age to the photosynthetic machinery or a disruption in the

Environmental Science and Pollution Research
balance of photosynthetic and respiratory processes (Xie
etal. 2019). For the outside CO2 levels, the initial increase
in outside CO2 with gamma-ray doses of 1Gy and 3Gy sug-
gests that low-to-moderate levels of gamma radiation may
stimulate processes that result in higher emissions of CO2
into the environment. This could be due to radiation-induced
changes in cellular metabolism, leading to an upsurge in CO2
production (Jaiswal etal. 2023). The continuous increase in
outside CO2 with escalating gamma-ray doses up to 3Gy
might indicate a direct correlation between gamma radia-
tion and CO2 release, at least within this range. This could
be linked to a dose-dependent increase in metabolic activity,
stress responses, or alterations in normal cellular functions
due to radiation exposure. However, the subsequent decline
in outside CO2 levels at a gamma-ray dose of 5Gy suggests
a possible threshold effect (Bera etal. 2022). This drop could
indicate that the system’s ability to cope with radiation stress
is overwhelmed, leading to a slowdown in metabolic activity
or damage to cellular components in CO2 production. The
system might also use stress-mitigation strategies that reduce
metabolic activity to conserve energy (Polutchko etal.
2022). It is also possible that this threshold effect represents
an automated mechanism where the system tries to main-
tain balance under increased radiation stress. The dynamic
response to varying doses of gamma radiation reflects the
intricate balance between stress adaptation and damage in
plants, with significant implications for understanding plant
responses to environmental stressors (Mannucci etal. 2020).
Gamma-ray dosages and net photosynthetic rate (Pn)
showed a surprising association. The positive connection
indicates that larger gamma-ray doses may increase photo-
synthetic activity in the examined system, resulting in higher
net photosynthesis rates. This could be due to gamma radia-
tion’s hormetic effects on the photosynthetic system. Low to
moderate radiation exposure may trigger adaptive responses
in the photosynthetic machinery, improving efficiency and
net photosynthesis (Babina etal. 2020). This phenome-
non might involve alterations in photosynthetic pigments,
enzyme activity, or energy harvesting mechanisms, adapting
the system to utilize the energy from gamma rays effectively
(Hussein 2022). Even after a 5-Gy gamma-ray exposure, the
net photosynthesis rate keeps rising, suggesting the exam-
ined system has a higher tolerance to radiation. However,
a threshold effect may occur at this level, causing photo-
synthetic activity to plateau or diminish, possibly due to
radiation-induced damage outweighing adaptive responses.
Photosynthetically active radiation (PAR) increased signifi-
cantly during exposure to 1 and 3Gy gamma-ray doses. This
suggests that mild to moderate radiation exposure may posi-
tively influence the photosynthetic system, likely owing to a
hormetic impact (Song etal. 2021). Radiation stress at these
concentrations may stimulate adaptive mechanisms, enhanc-
ing photosynthesis and increasing PAR. This response could
be due to the activation of protective pathways that enhance
light energy utilization under stress conditions (Wang etal.
2022). However, the considerable decline in PAR at a 5-Gy
gamma-ray dosage suggests a threshold beyond which the
system may struggle to maintain improved photosynthetic
activity. This decline could result from extensive radiation-
induced damage to the photosynthetic machinery, reducing
light absorption and overall photosynthesis (Ahmed etal.
2021). Damage to photosynthetic pigments or structural
components under high radiation stress may critically affect
PAR and diminish photosynthetic performance (Duarte-
Sierra etal. 2020). These findings imply that while gamma
radiation can enhance photosynthetic activity at low to
moderate doses, it may become detrimental at higher doses,
affecting key photosynthetic parameters (Duarte-Sierra etal.
2020).
Our findings provided intriguing insights into gamma
radiation’s effects on plants’ anthocyanin production, dem-
onstrating a complex, non-linear relationship. The decrease
in anthocyanin content observed with a gamma radiation
dose of 1Gy, as compared to the control group, suggests
that low levels of gamma radiation may negatively impact
anthocyanin synthesis. This outcome could be attributed to
radiation-induced disruptions in the biosynthetic pathways
involved in anthocyanin production or alterations in the
regulatory mechanisms that control these pathways (Jaiswal
etal. 2021). Conversely, an increase in anthocyanin content
was noted when the gamma radiation dose was raised to
3Gy, indicating that intermediate levels of gamma radia-
tion may stimulate anthocyanin production. This increase
might be a stress response, as anthocyanins are known to
serve as protective compounds in plants under various stress
conditions, including radiation exposure (Li etal. 2022). The
production of anthocyanins in response to radiation might
help plants mitigate oxidative damage caused by gamma
rays (Akbari etal. 2022). However, a significant reduction
in anthocyanin content at the highest tested gamma radia-
tion dose (5Gy) could signify a threshold effect. Beyond a
certain level of radiation exposure, the detrimental effects
on the plant’s physiological processes and overall health
might outweigh the protective benefits, leading to a decline
in anthocyanin production (Neale etal. 2023). This could
also indicate more severe radiation-induced damage to the
pathways or regulatory mechanisms involved in anthocyanin
production (Gao etal. 2023a). These results underscore the
potential utility of gamma radiation in modulating antho-
cyanin content in plants, with potential applications in plant
breeding and agricultural practices, especially for enhancing
anthocyanin content in specific crops.
The initial increase in flavonoid content observed at a
gamma-ray dose of 1Gy might result from a stimulatory
effect of low-level radiation exposure, known as hormesis
(Volkova etal. 2022). The modest increase in flavonoid

Environmental Science and Pollution Research
content supports this suggestion, approximately 7.7% higher
than the control group. However, the observed reduction in
flavonoid content at higher gamma-ray doses (3 and 5Gy)
indicates a contrasting response. This decline could be
caused by the detrimental effects of higher radiation doses
on the biosynthetic pathways responsible for flavonoid pro-
duction, or it might reflect a shift in resource allocation
within the plant due to the radiation-induced stress (Fer-
reyra etal. 2021). A decrease in flavonoid content at higher
doses, despite an initial increase at a lower dose, suggests a
threshold dose for radiation exposure. Beyond this thresh-
old, the negative impacts of radiation might surpass any
potential stimulatory effects (Jones etal. 2020). These find-
ings emphasize the importance of understanding the dose-
dependent effects of gamma radiation on plant physiology,
particularly regarding the production of beneficial secondary
metabolites like flavonoids (Sharma etal. 2022).
The above results suggest a multifaceted impact of
gamma radiation on plants’ peroxidase (POD) activity. In the
initial exposure stage at 1Gy, the gamma radiation decreases
POD activity, potentially due to radiation-induced cellular
damage, which might alter the enzymatic structure or func-
tion (Ghasemi etal. 2019). However, the observed increase
in POD activity at a 3-Gy dose indicates a possible compen-
satory response or adaptation mechanism in the plant, likely
triggered by the induction of stress-responsive pathways
(Rajput etal. 2021). This increase in activity might also be
related to the plant’s inherent defense mechanism, as POD
plays a critical role in plant responses to various stressors,
including radiation stress (Esnault etal. 2010). Conversely,
at a higher dose of 5Gy, a decrease in POD activity was
noted again. This reduction might be hypothesized as a result
of substantial cellular damage caused by higher doses of
gamma radiation, which could exceed the plant’s capacity
for compensation, leading to a decline in enzyme activity
(Latorre etal. 2010).
Additionally, prolonged exposure to high radiation levels
might lead to a depletion of substrates necessary for POD
activity, thereby reducing enzymatic function (Ghasemi
etal. 2019). These findings, indicating a biphasic response
pattern to gamma radiation, highlight plants’ delicate bal-
ance under radiation stress (Georgieva and Vassileva 2023).
They underscore the importance of optimizing radiation
exposure levels to maintain POD activity, which is crucial
for the overall health and resilience of the plant.
The observed dose-dependent relationship between
gamma-ray exposure and the expression of thymol synthase
genes provides exciting insights into the regulation of thymol
synthesis in plants. To discuss potential biological mecha-
nisms underlying these observed dose-dependent effects, it is
noteworthy that at low doses of gamma radiation (1Gy), the
induction of thymol synthase gene expression might result
from a stress-induced signaling response. This response is
part of the plant’s sophisticated defense mechanisms devel-
oped to counteract environmental stressors, including radia-
tion (Jit etal. 2021; Hoque etal. 2020). Activating signal
transduction pathways involved in gene expression regu-
lation under mild stress could lead to the upregulation of
thymol synthase gene expression and increased thymol pro-
duction. When exposed to higher gamma-ray doses (3Gy),
the significant increase in thymol synthase expression aligns
with the concept of hormesis, where moderate stress levels
trigger more pronounced adaptive responses, enhancing thy-
mol production (Jacobo-Velázquez etal. 2022; Pandey etal.
2023). Thymol’s antimicrobial and antioxidant properties
highlight its role in mitigating cellular damage and maintain-
ing homeostasis under stress conditions (Rathod etal. 2021).
However, at even higher doses (5Gy), the slight decrease
in thymol synthase expression could be explained by the
plant’s strategy to manage excessive stress, thereby conserv-
ing energy and allocating resources toward repair and stress
mitigation (Xie etal. 2022; Katiyar etal. 2022; Mahajan
etal. 2020). Exploring hypotheses or mechanisms for the
threshold effects observed, the data suggest a nuanced inter-
action between gamma-ray dosage and plant physiological
responses. Beyond a certain radiation threshold, the plant’s
adaptive mechanisms seem overwhelmed, reducing benefi-
cial responses such as thymol production. This highlights
a critical balance point where radiation transitions from a
growth stimulant to a growth inhibitor. Aligning these find-
ings with existing literature on plant stress responses and
secondary metabolite biosynthesis (Reviewer Comment 3),
the results are consistent with the known impacts of environ-
mental stress on secondary metabolite pathways in plants.
The modulation of thymol production in response to varying
levels of gamma radiation indicates the dynamic nature of
these biosynthetic pathways under stress conditions (Farooq
etal. 2019). In terms of practical applications and future
research directions, these findings could inform the develop-
ment of gamma-ray treatments to optimize the production
of valuable secondary metabolites like thymol in medici-
nal and aromatic plants. This has significant implications
for enhancing the quality and yield of these compounds for
pharmaceutical and agricultural applications.
This study illuminates the intriguing correlation between
gamma-ray doses and the expression of the γ-terpinene
synthase (TPS) gene, pivotal for γ-terpinene production.
The data underscores that gamma radiation exposure pro-
motes the expression of the TPS gene, thereby amplifying
γ-terpinene production. Discussing the potential biological
mechanisms underlying these dose-dependent effects, it is
observed that at a gamma-ray dose of 1Gy, there is a nota-
ble increase in TPS gene expression, suggesting that even
marginal doses of gamma rays are effective in enhancing
γ-terpinene production. This enhancement is likely tied to
activating signaling pathways crucial to stress responses

Environmental Science and Pollution Research
(Ghasemi etal. 2019). Gamma radiation, known for induc-
ing oxidative stress and DNA damage, leads plants to trigger
a variety of defense mechanisms, including the modulation
of gene expression (Belykh etal. 2022). The increase in TPS
gene expression at this dose can be viewed as a plant’s defen-
sive strategy to augment γ-terpinene production, a com-
pound recognized for its antioxidant and antimicrobial prop-
erties, thereby strengthening the plant’s resilience against
radiation-induced stress (Zhang etal. 2019). As the gamma-
ray dose is increased to 3Gy, a significant upregulation in
TPS gene expression is observed. This marked increase,
nearly 274% greater than the 1Gy dose, suggests a robust
response to the higher radiation levels. This response might
be attributed to activating specific transcription factors and
signaling pathways that regulate secondary metabolite bio-
synthesis genes (Hussain etal. 2021). The enhanced expres-
sion level of the TPS gene at this dose underlines the plant’s
adaptive strategy to cope with moderate levels of radiation-
induced stress, boosting γ-terpinene production as part of
this adaptive response. Exploring the threshold effects, it is
particularly intriguing that upon increasing the gamma-ray
dose to 5Gy, a dramatic surge in TPS gene expression is
observed despite the potential for DNA damage and cel-
lular disruption at such high radiation levels (Tariverdiza-
deh etal. 2023). This suggests a complex interplay between
radiation dose and gene expression, where higher doses of
radiation may invoke additional stress-responsive genes and
pathways, potentially leading to amplified TPS gene expres-
sion. However, the slight dip in gene expression in certain
instances at this high dose could indicate a threshold beyond
which the adverse effects of radiation begin to outweigh the
stimulatory effects. The study contributes valuable insights
into the complex dynamics of gene expression regulation
under varying levels of environmental stress. The increase
in γ-terpinene production in response to different doses of
gamma radiation aligns with known patterns of secondary
metabolite modulation under stress conditions. These find-
ings could inform strategies for optimizing the production
of valuable compounds like γ-terpinene in plants using con-
trolled gamma-ray exposure. This has potential applications
in industries such as flavoring, fragrance, and pharmaceuti-
cals, where γ-terpinene is a crucial component.
The observed augmentation in carvacrol synthase activity
following a gamma-ray dose of 3Gy, in comparison to the
control group, highlights a stimulatory effect of moderate
gamma-ray exposure on this enzyme. Carvacrol synthase,
critical for carvacrol biosynthesis, is vital due to carvacrol’s
antimicrobial and medicinal properties (Rabiei etal. 2018).
This enhanced enzyme activity suggests that gamma rays at
a dose of 3Gy can stimulate carvacrol synthase, potentially
leading to increased carvacrol production. This phenomenon
may be attributed to the activation of specific signaling path-
ways or transcriptional regulators that positively modulate
the expression or activity of carvacrol synthase, a hypothesis
supported by existing literature on plant responses to envi-
ronmental stressors (Belykh etal. 2022; Song etal. 2021).
Exploring the potential biological mechanisms underlying
these dose-dependent effects, the gamma rays, known for
inducing DNA damage, trigger various cellular responses,
including gene expression changes. The moderate dose of
3Gy stimulates mechanisms favoring carvacrol synthase
activity, which aligns with the theory of hormesis, where
moderate stress levels can benefit biological systems (Volk-
ova etal. 2022). However, a notable decline in carvacrol syn-
thase activity at a higher gamma-ray dose of 5Gy suggests
a threshold effect (Reviewer Comment 2). At this elevated
dose, the detrimental impact of gamma rays might surpass
the stimulatory effects, leading to reduced carvacrol produc-
tion. This could be due to extensive DNA damage and dis-
ruption of cellular processes, adversely affecting the stability
or functionality of carvacrol synthase (Jaiswal etal. 2023;
Li etal. 2022).
The discernible influence of varying gamma-ray doses on
the production of thymol and carvacrol provides valuable
insights into the physiological reactions within the tested
samples. Compared to the control group, the observed
increase in thymol content upon exposure to a 1-Gy gamma-
ray dose suggests a stimulatory effect of gamma rays on thy-
mol production. This increment aligns with the hypothesis
that low-dose gamma radiation can activate enzymes critical
in thymol biosynthesis, leading to enhanced production. This
phenomenon may be attributed to the modulation of gene
expression and metabolic pathways induced by gamma rays,
a concept supported by current research in plant molecu-
lar responses to environmental stressors (Gao etal. 2023b;
Volkova etal. 2020). Further, the significant augmenta-
tion of thymol content at a 3-Gy dose highlights the dose-
dependent nature of this response, suggesting heightened
enzymatic or transcriptional activity in thymol biosynthesis
pathways at moderate gamma-ray doses (Rabiei etal. 2018;
Qi etal. 2014). However, the decrease in thymol content at a
5-Gy dose suggests a threshold effect, where higher levels of
gamma radiation may negatively impact thymol production,
possibly due to excessive stress or damage to biosynthetic
pathways.
Similarly, the increase in carvacrol content at a 1-Gy
gamma-ray dose indicates a stimulatory effect of gamma
rays on its production. This observation is consistent with
the theory that gamma rays can enhance the activation or
upregulation of enzymes involved in carvacrol biosynthesis,
leading to increased production of this bioactive compound.
However, the subsequent reduction in carvacrol content at a
5-Gy dose implies a threshold effect or a shift in the plant’s
physiological response to higher doses of gamma radiation,
potentially leading to decreased biosynthetic efficiency
(Volkova etal. 2020). These findings, reflecting a complex

Environmental Science and Pollution Research
interplay between gamma radiation, DNA damage, cellular
responses, and secondary metabolite synthesis, offer essen-
tial insights into optimizing gamma-ray doses for enhancing
the production of valuable compounds like thymol and car-
vacrol. They highlight the critical role of radiation in mod-
ulating plant biochemistry and underscore the importance
of considering genetic and metabolic traits in response to
gamma-ray exposure. Such understanding is vital for future
research and practical plant bioactive compound production
applications.
The observed increase in γ-terpinene concentration at a
1-Gy gamma-ray dose compared to the control group sug-
gests that gamma-ray exposure can enhance its synthesis.
γ-Terpinene, a key component in essential oils used in the
scent and flavoring industry, shows a response to gamma
radiation, hinting at the activation of specific biosynthetic
pathways under radiation exposure (Du etal. 2023; Rahman
etal. 2023). This increase is likely due to the stimulation
of signaling pathways and transcriptional regulators, which
play a pivotal role in the biosynthesis of γ-terpinene. At a
moderate gamma-ray dose of 3Gy, the significant increase
in γ-terpinene content further supports the theory of radia-
tion hormesis, where moderate stress levels stimulate biolog-
ical responses, enhancing secondary metabolite production
(Ulukapi and Nasircilar 2022). The role of γ-terpinene and
similar monoterpenes in plant stress responses, particularly
their antioxidative properties, aligns with existing litera-
ture on plant adaptation to environmental stress (Hsouna
etal. 2019). By acting as antioxidants, these compounds
may offer protective effects against oxidative stress, con-
tributing to plant resilience under such conditions. How-
ever, the decrease in γ-terpinene content at a higher dose of
5Gy introduces a complex dynamic, suggesting a threshold
effect where excessive radiation might harm its biosynthesis.
High gamma-ray doses could lead to physiological stress
or damage, adversely impacting the enzymatic pathways
and cellular processes involved in γ-terpinene production.
This nonlinear response highlights the importance of care-
fully calibrating gamma-ray doses to optimize γ-terpinene
production, balancing the stimulatory effects at lower doses
against the potential negative impacts at higher doses. These
findings provide a nuanced understanding of how gamma-
ray treatments can modulate the levels of γ-terpinene, a valu-
able compound in various industries. They underscore the
potential of using gamma-ray irradiation in altering the com-
position of essential oils, with implications for enhancing the
production of γ-terpinene in commercially valuable plants.
This understanding of dose-dependent responses and the bal-
ance between stimulation and stress in γ-terpinene biosyn-
thesis offers valuable insights for future research and practi-
cal applications in plant secondary metabolite production.
The correlation analysis in this study underscores the
intricate relationships between morphological traits in
Thymus vulgaris under low-dose gamma radiation, offering
insights into monoterpene biosynthesis and plant adapta-
tions. The strong positive correlation between total fresh
weight (TFW) and shoot fresh weight (SFW) reinforces the
notion that low-dose gamma radiation boosts the growth
and development of Thymus vulgaris, signifying a positive
impact on shoot growth and biomass accumulation. This
effect likely results from the stimulation of metabolic and
cellular growth pathways under gamma radiation (Qi etal.
2014). A moderate positive correlation between TFW and
root length (RL) points toward enhanced root development
under low-dose gamma radiation, critical for nutrient uptake
and plant stability. This indicates that gamma radiation may
stimulate root growth, a vital aspect of plant fitness, possibly
by activating cell division and elongation in the root meris-
tem (Rongsawat etal. 2021; Volkova etal. 2020). Addition-
ally, the correlation between TFW and the number of nodes
per plant (NN) suggests that gamma radiation influences
vegetative growth and branching in Thymus vulgaris. An
increase in nodes reflects enhanced shoot branching, likely
driven by the modulation of hormonal pathways and bud
development processes under gamma radiation (Volkova
etal. 2022). The positive correlation between RL and SFW
indicates a harmonious relationship between shoot and root
growth, essential for maintaining a balance in biomass dis-
tribution. This suggests that gamma radiation facilitates
growth in both parts of the plant through growth-promot-
ing hormones and signaling pathways (Zhang etal. 2022;
Duarte etal. 2023). Notably, anthocyanins and flavonoids
exhibit a strong positive correlation, hinting at co-regula-
tion or shared biosynthetic pathways. These compounds,
known for their antioxidant properties, may be stimulated
by low-dose gamma radiation as a defense against oxidative
stress (Ghasemi etal. 2019). Polyphenol oxidase (PPO) and
peroxidase (POD) also show a strong positive correlation,
indicating coordinated enzymatic activities in response to
radiation stress. This supports the idea that gamma radiation
may enhance the activities of these enzymes, bolstering the
plant’s defense mechanisms (Harish etal. 2023; Katiyar etal.
2022). Moreover, the strong correlations between Thymol
synthase (CYP71D178), carvacrol synthase (CYP71D180),
and their respective products suggest that gamma radiation
enhances the biosynthesis of these monoterpenes. This could
be part of the plant’s adaptive mechanism to radiation stress,
potentially increasing the production of thymol and carvac-
rol for protection (Rabiei etal. 2018; Tariverdizadeh etal.
2023; Akhi etal. 2021). These correlations, particularly
between thymol and carvacrol with traits like PPO, POD,
and flavonoids, imply shared metabolic pathways and inter-
actions among these traits in response to gamma radiation.
The findings suggest that gamma radiation may stimulate the
biosynthesis of thymol and carvacrol as an adaptive response
to radiation-induced stress, providing valuable insights for

Environmental Science and Pollution Research
future research in plant physiology and secondary metabolite
production (Jit etal. 2021).
The cluster analysis results offer valuable insights into the
differential impacts of gamma radiation doses on thyme. The
clustering of the control and 1Gy treatment together suggests
that a low dose of 1Gy gamma radiation may not significantly
affect the physiological state of thyme compared to untreated
controls. This observation implies that the studied physiologi-
cal traits are either not highly sensitive to this radiation level
or that the plant’s cellular repair mechanisms efficiently coun-
ter the damage caused by such low-level radiation exposure
(Nawkar etal. 2013). In stark contrast, the 5-Gy treatment,
forming a distinct cluster, displayed the lowest values across
all traits. This could indicate that a higher radiation dose of
5Gy is detrimental to thyme, possibly impairing its growth
and overall health. Such adverse effects may be attributed to
the harmful impact of radiation at this higher dosage, poten-
tially resulting in cellular damage, metabolic disturbances,
and inhibited growth processes (Jan etal. 2012). This find-
ing supports the idea that the 5-Gy dose exceeds the plant’s
defensive and reparative capabilities threshold, leading to a
noticeable decline in the measured traits (Kovacs and Keresz-
tes 2002). Conversely, the 3Gy treatment, which formed its
cluster, exhibited the highest values for nearly all traits. This
intriguing outcome suggests a hormetic response, where a
moderate radiation dose seems to confer beneficial effects on
thyme. Hormesis, a response in which moderate stress induces
a positive effect, could be at play, indicating that the 3Gy dose
might have activated the plant’s defense or repair mechanisms.
This response could be seen as an adaptive physiological strat-
egy to moderate stress, enabling the plant to withstand and uti-
lize it to enhance its overall functionality (Duarte-Sierra etal.
2020; Ellis and Del Giudice 2019). These findings highlight a
dose-dependent relationship between gamma radiation and its
physiological effects on thyme. The effects of radiation range
from negligible at lower doses to harmful at higher doses,
with potential benefits observed at moderate doses. This
emphasizes the importance of carefully optimizing gamma
radiation doses in agricultural treatments to achieve desired
outcomes while avoiding adverse effects (Alothman etal.
2009; Volkova etal. 2022).
Conclusion
In conclusion, the findings of this study shed light on
the profound influence of low-dose gamma radiation on
Thymus vulgaris’ metabolic processes, specifically in the
biosynthesis of characteristic monoterpenes such as thy-
mol and carvacrol. The strong positive correlations found
between enzymes like Thymol synthase (CYP71D178)
and Carvacrol synthase (CYP71D180) and their respec-
tive end products indicate that low-dose radiation could
potentially stimulate their expression or activity, thereby
enhancing the production of these vital secondary metabo-
lites. This implies an adaptive mechanism in which the
plant induces the production of these compounds as a
protective response against radiation stress. Further, the
study also uncovers possible links and interactions among
various traits and metabolic pathways enriched by expo-
sure to gamma radiation. While the correlation between
γ-terpinene synthase and other traits is relatively weak, a
promising positive correlation with Thymol suggests an
exciting avenue for future research. These insights contrib-
ute to our understanding of the plant’s molecular response
to radiation and pave the way for a deeper exploration of
the regulation of monoterpene biosynthesis under stress
conditions. The potential of low-dose gamma radiation
to trigger specific metabolic responses, as evident in this
study, opens up new possibilities for enhancing the pro-
duction of beneficial plant secondary metabolites. The
study also presented an intricate exploration of the impacts
of varying gamma-ray doses on various plant attributes.
It unearthed a complex relationship between gamma-ray
exposure and the examined parameters. The data revealed
a general trend of initial increases in these parameters
upon exposure to low to moderate gamma-ray doses, fol-
lowed by decreases at higher doses. This suggests a pos-
sible threshold effect where the parameters start to decline
beyond a certain level of gamma-ray exposure.
Interestingly, the net photosynthesis rate deviated from
this trend, displaying a continuous increase even at higher
doses, suggesting enhanced photosynthetic activity with
increasing gamma-ray doses. This investigation adds valu-
able insights to the body of knowledge on the impact of
gamma rays on plant development and metabolic processes.
It indicates the potential of gamma radiation as a tool for
plant growth modulation. These findings, however, also sug-
gest that the effects of gamma radiation on plants are intri-
cate and dose-dependent. Hence, while gamma rays could
potentially be harnessed for beneficial purposes in plant
science, careful consideration must be given to the dosage
applied to avoid detrimental effects.
Author contribution FS, SS, and MK proposed the idea of the paper.
MK and SS prepared the manuscript and figures. FS, SS, MK, and AG
wrote the manuscript. All authors read and approved the final version
of the manuscript.
Funding This study was supported by grants from the National
Institute of Genetic Engineering and Biotechnology (project code—
010214-II-804, entitled “Increasing Thymol Production in Thymus vul-
garis by Optimizing the Induction Method with Low Doses of Gamma
Rays”).
Data availability The datasets used or analyzed during the current
study are available from the corresponding author upon reasonable
request.

Environmental Science and Pollution Research
Declarations
Ethical approval Not applicable.
Consent to participate Not applicable.
Consent for publication Not applicable.
Conflict of interest The authors declare no competing interests.
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