Non-destructive methods of
analysis are increasingly being used to study the vital state of plants and the
content of various metabolites [1-3]. These methods allow for the measurement
of substance content without damaging or destroying plant organs, which is
particularly important when multiple measurements are required. Spectral
methods enable the evaluation of various morpho-physiological and biochemical
indicators of plants, as well as their vital state, without the need for
time-consuming sample preparation in both field and laboratory conditions in
response to various stress factors. [4-7].
However, the main issue
lies in the frequent evaluation of only well-known metabolites, without taking
into account those that can be detected at other wavelengths.
Additionally, the analysis often focuses on the
content of entire groups of substances, such as flavonoids [8], carotenoids
[9], etc. Therefore, visualization of the vital state through spectral profiles
can provide a more comprehensive picture of plant adaptation to stress factors.
The object selected for
the pilot study of spectral profiles was the pedunculate oak (Quercus robur L.), known for its high drought resistance and being one of the main woody
species used for agroforestry and protective afforestation in southern Russia
[10, 11]. Therefore, conducting a spectral analysis of the leaf blade can
provide a comprehensive understanding of how
Q. robur
responds to
drought.
The aim of this study was
to conduct a pilot study to evaluate the spectral profiles of
Q. robur
leaf blades under simulated drought conditions.
To model experimental drought, 5-6
month-old
Q. robur
seedlings without signs of phytopathology were used.
The seedlings were divided into three groups: a control group, a group with
watering and a group without watering (drought), with 15 seedlings in each
group. Spectral diagnostics of
Q. robur
leaves was performed at 0
(control), 168 (one week) and 336 (two weeks) hours. After measuring the
spectral parameters of the leaves of the control group, the temperature in the
experimental room was set to 30˚C.
A portable spectroradiometer SpectroPen
SP110 Uvis (Photon Systems Instruments, Czech Republic) [12], which registers
radiation in the wavelength range from 340 nm to 790 nm, was used to obtain
spectral profiles of the leaf blades of Q
. robur
seedlings under study.
Optical radiation was recorded from the abaxial side of the leaf blade next to
the midrib. The reference radiation was sunlight, with the optical path passing
through the registration site on the leaf blade. A specialized program
SpectraPen v.1.1.0.10 (Photon Systems Instruments, Czech Republic) was used to
export the primary spectral profile data of
Q. robur
leaf blades. The
obtained data in the .scv format were converted to Excel spreadsheet format
(Microsoft, USA) and used for further statistical processing.
The content of the sum of chlorophylls,
flavonols, and anthocyanins in μg/cm2)
in the leaves epidermis
was measured using a plant analyzer Dualex Scientific+ ("Force-A",
France).
The formula below was used to construct
spectrograms of radiation absorption:
,
where A is the optical density coefficient,
I0
is the reference radiation intensity, I is the measured radiation
intensity.
Quantitative data were analyzed using
Statistica 12.0 software (StatSoft Inc., USA) [13]. The indicators used to
evaluate nonparametric samples in biological studies were calculated: normality
of value distribution, median [1st quartile, 3rd quartile], and the
significance of sample differences was analyzed. To determine the differences
between two independent samples, the Mann–Whitney test was used at a
significance level of p<0.05.
As a result of the study,
spectrograms of radiation absorption of
Q. robur
leaf blades were
obtained in the wavelength range of 340-790 nm under watering and drought
conditions at three time intervals: 0 (control), 168 (one week), and 336 (two
weeks) hours. The spectrograms of radiation absorption had two peaks: a higher
and narrower peak in the range of 415-480 nm, as well as a lower and broader
peak in the range of 481-635 nm (Fig. 1). The absorption peaks were highest in
the control group and decreased in the experimental groups. Moreover, the most
significant differences in absorption values were observed in the wavelength
ranges corresponding to the aforementioned peaks, while differences outside
these ranges were minimal. This indicates that drought stress affected the
leaves ability to absorb light. At the same time, the leaf blades changed over
time under the influence of stress factors (Fig. 2A). In this case,
spectrograms help to track changes in the vital state of
Q. robur
leaf
blades.
Fig. 1. Spectrograms of
radiation absorption of
Q. robur
leaf blades under simulated drought
conditions
Based on the literature
data, it can be assumed that the first peak represents the peak of chlorophylls
and carotenoids [14-16]. The second broad peak can be formed by anthocyanins
[17, 18]. The peak of flavonols is not observed. This may be due to the fact
that the device does not record values at all wavelengths for flavonols [19], as
well as the possibility that the peak for flavonols is small and may be
overlapped by other metabolites. Subsequently, to study the vital state of the
leaf blade (Fig. 2A) and its connection to the spectral profile of radiation
absorption (Fig. 2B, 2D, 2F, 2H), spectra were taken at wavelengths of 415-480
nm and 481-635 nm.
Fig. 2. Vital state,
pigment content and the spectrograms of radiation absorption of
Q. robur
leaf
blades under simulated drought conditions: A – the vital state of
Q. robur
leaf blades; B – the first peak of the spectrograms of radiation absorption
after one week of the experiment; C – comparison of the sum of chlorophylls
content in leaf blades; D – the first peak of the spectrograms of radiation
absorption after two weeks of the experiment; E – comparison of the flavonol
content in leaf blades; F – the second peak of the spectrograms of radiation
absorption after one week of the experiment; G – comparison of anthocyanin
content in leaf blades; H – the second peak of the spectrograms of radiation
absorption after two weeks of the experiment
The
Q. robur
leaf
blades were visually evaluated. One and two weeks after the beginning of the
experiment, it was observed that the leaf blades from the watering group did
not differ much from the control group. At the same time, leaf blades from the
drought group showed precise signs of wilting.
Figures 2B and 2G show a
significant difference in absorption peaks (2.5-3 times) between the control
and experimental groups in the 415-480 nm range. Two weeks after the beginning
of the experiment, the spectral values indicate a more pronounced difference in
absorption (1.5 times) between the watering and drought groups.
Figures 2F and 2H also
demonstrate significant differences between the groups in the 481-635 nm range.
Compared to the control peak, the absorption is 2-3 times lower in the
experimental groups. In the graphs of the first week, absorption is slightly
higher in the experimental drought group than in the watering group. However,
this situation changes after two weeks.
Statistically
insignificant differences are observed in the watering and drought graphs in
the first week at the following wavelengths: 416, 435, 436, 439-445, 452, and
470 nm. Additionally, the differences between the control and drought groups in
the second week are not significant at wavelengths 434-435, 472-473, and
476-477 nm. In Figures 2E and 2H, no statistically significant differences are
observed between drought and watering in the first week at wavelengths 517 nm
and 593 nm, and between control and drought at 607, 608 nm. In the second week,
p>0.05 between watering and drought groups at wavelengths 483-485, 504,
513-514, 544, 587, 592-593, 597, 602, 605-606, 615, 619, 621-622, 624, 627-628,
and 634 nm.
Figures 2Ñ, 2E, and 2G
present diagrams of the sum chlorophylls, flavonol, and anthocyanin content,
respectively. In the control group, the chlorophyll content was 14 µg/cm².
After one week, the chlorophyll content increased by 1.5-2 times in the
experimental groups, with the drought-simulated group showing higher
chlorophyll levels compared to the watering group. After another week, the
chlorophyll content in the experimental groups slightly decreased compared to
the previous week (18 µg/cm² and 21 µg/cm² for the watering and
drought groups, respectively). The chlorophyll level in the drought group was
slightly higher than in the watering group. It is hypothesized that plants
began to produce more chlorophyll in response to stress. One possible reason
for this increase may be a stress-induced response that leads to an increase in
chlorophyll production for a certain period of time, which allows maintaining photosynthesis
levels and ensuring survival [20, 21].
It is known that the
content of flavonols can vary under different stress conditions [22], including
an increase under drought stress [23-25]. Compared to the control, the content
of flavonols in the experimental groups slightly increased (p < 0.05). The
content of anthocyanins decreased by approximately 1.5-2 times, with the
drought group showing lower anthocyanin content compared to the watering group.
After two weeks, the content of anthocyanins increased compared to the first
week. Presumably, this is associated with the plant adaptation to stress, in
this case, an increase in the level of anthocyanins.
Statistically significant
differences in the sum of chlorophylls were found between the first and second
weeks of watering, as well as between the second week of watering and the
second week of drought (p < 0.05) (Fig. 2B). The difference in flavonol
content was significant for all groups (p < 0.05), except for values between
the watering groups in the first and second weeks (Fig. 2D). In the graph
showing anthocyanin content, values between different drought and watering
groups are statistically significant (p < 0.05). Statistically insignificant
differences from the control group were found only between the flavonol content
in the first week of drought and the second week of watering.
Thus, the changes in
pigment content caused by the adaptation of
Q. robur
seedlings to stress
led to alterations in the spectrograms of the corresponding pigments. However,
due to the use of an uncontrolled radiation source, the obtained spectrograms
may differ from the reference spectrograms of the corresponding pigments
acquired using invasive methods [26]. According to the Bouguer–Lambert–Beer
law, the obtained optical density spectrograms represent the sum of radiation
absorption not only by the pigments but also by other metabolites and chemicals
present in the leaf blade, as well as a result of scatter of solar radiation by
the plant tissue of the leaf blade. The absorption spectrum of chlorophyll and
other substances can be affected by environmental factors, such as pH,
temperature, the presence of other molecules, and changes in chlorophyll
structure due to stress [27, 28]. These factors can cause the absorption peak
to shift towards the blue region and decrease in the red region.
During the simulation of
experimental drought in
Q. robur,
changes in the absorption spectrograms
of leaf blades and the quantitative content of pigments were recorded, which
may be related to the impact of water deficiency on the physiological processes
of plants. The observed differences in the spectrograms indicate changes in the
content of pigments and other metabolites, as well as structural alterations in
the leaf caused by drought.
It is hypothesized that
the first peak in the spectrogram corresponds to the peak of chlorophylls and
carotenoids, while the appearance of the second broad peak indicates the
presence of anthocyanins, and the peak for flavonols was not observed.
Differences in absorption peaks were observed between control and experimental
groups in the wavelength ranges of 415-480 nm and 481-635 nm. In the first
week, absorption was slightly higher in the drought group compared to the
watering group. Two weeks after the experiment commenced, the differences in
absorption between the groups became more pronounced: compared to the control,
the peak in the experimental groups was 2-3 times lower.
It is known that plants
begin to produce more chlorophyll and other substances to enhance
photosynthesis efficiency and ensure survival under stress conditions, such as
drought. In the experimental groups, an increase in the chlorophyll content was
observed. In the control group, the chlorophyll content was 14 µg/cm².
After one week, the chlorophyll content increased by 1.5-2 times, and after
another week it slightly decreased (18 μg/cm2
in the watering
group and 21 μg/cm2
in the drought group). In the experimental
groups, there is an insignificant increase in the flavonol content compared to
the control group. In most cases, the values of absorption peaks have
statistically significant differences (p<0.05). At the same time, the
anthocyanin content decreased by approximately 1.5-2 times (p<0.05) in all
experimental groups compared to the control. The lowest anthocyanin content was
observed in the drought group compared to the watering group after one week of
the experiment. However, in the second week, the anthocyanin content increased,
which may indicate plant adaptation to stress and the recovery of the
anthocyanin production level.
During the pilot study, it
was revealed that changes in the spectrograms of radiation absorption of
Q.
robur
leaf blades are associated with possible alterations in the content
of a wide range of metabolites such as chlorophyll, carotenoids and
anthocyanins. It was found out that the difference in absorption peaks between
the control and experimental groups became more pronounced over time,
indicating the impact of drought conditions. In the control group, the
spectrograms of radiation absorption demonstrated the highest ability to absorb
radiation by leaves, while in the experimental drought groups it decreased.
Simultaneously,
Q. robur
leaf blades underwent morphological changes
under stress conditions.
It can also be concluded
from the results of the study that exposure to stress factors, such as drought,
contributes to an increase in the plant chlorophyll content, enhancing
photosynthesis efficiency and contributing to their survival. The flavonol
content slightly increases, which may be due to the absence of
phytopathologies. At the same time, the anthocyanin content decreases under
stress conditions. However, their recovery occurs over time, indicating plant
adaptation to stress conditions.
Thus, the spectrograms
allow tracking changes in the viability of
Q. robur
leaf blades, most
noticeable in the range corresponding to the absorption peaks on the obtained
spectrograms. Further investigations are needed to study spectral profiles and
their connection to plant adaptation in response to stress factors,
particularly, drought. The obtained preliminary results can be used in the
future to develop methods for evaluating vital state, physiological and
biochemical parameters and calculated indexes of woody plants in the field.
This research work
supported by the framework of the state task of the Ministry of Science and
Higher Education of the Russian Federation No. FNFE-2025-0013 “Search and
management of patterns of expression of forest and cultural plant genes
responsible for adaptation to environmental hazards and productivity”.
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