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https://doi.org/10.1007/s12633-023-02492-6

RESEARCH

Regulatory Role of Silicon on Photosynthesis, Gas‑exchange and Yield

Related Traits of Drought‑Stressed Lentil Plants
Sajitha Biju1 · Sigfredo Fuentes1 · Dorin Gupta1

Received: 29 March 2023 / Accepted: 26 April 2023

© Springer Nature B.V. 2023

Abstract
Purpose Drought is one of the most devastating abiotic constraints leading up to 60% lentil production losses. Silicon (Si)
application has been shown to be a promising drought stress management strategy in many Si accumulator crops. The present
study investigated whether Si can also help to mitigate drought stress in Si excluder crops like lentil.
Methods Experiments were conducted under both controlled and field conditions with selected drought susceptible and toler-
ant lentil genotypes, which were subjected to drought stress at the onset of the flowering. The measurements of chlorophyll
fluorescence, photosynthetic pigments, infrared thermal canopy temperature, leaf gas exchange parameters and yield traits
were analysed and compared to control treatments.
Results Silicon attenuated drought stress-mediated effects on light absorption of photosystem II (PSII) by increasing the
effective quantum yield of PSII photochemistry and photosynthetic pigments. Added Si maintained the cooler canopies of
stressed plants and mitigated negative effects of drought stress on gas exchange parameters. Increase in crop growth and
yield treated with Si under drought stress was primarily related to higher photosynthetic activity.
Conclusions Exogenous application of Si could be a potential on-farm and off-farm drought stress management strategy
for lentil plants.

Keywords Canopy · Chlorophyll · Fluorescence · Photosystem · Pigments · Yield

1 Introduction with severely damage photosynthetic traits, water relations

Drought stress inhibits a variety of physiological processes
in plants, where its most significant inhibitory effect can
be observed on photosynthesis [1]. Photosynthesis is essen-
tial to maintain growth and development of plants. Drought
stress causes a considerable damage to the PSII reaction cen-
tres in most plants leading to an imbalance between the PSII
photochemical activity and electron requirements for photo-
synthesis [2]. The photosynthetic systems in higher plants,
like lentil, are sensitive to drought stress [3]. Drought stress
can inhibit the assimilatory power of plants by stomatal limi-
tation (stomatal closure), non-stomatal limitation (destruc-
tion of photosynthetic membranes) and suppression of the
transport and distribution of photosynthetic assimilates from
source to sink [4]. Drought stress in lentil has been reported
* Dorin Gupta
dorin.gupta@unimelb.edu.au
1
School of Agriculture and Food, Faculty of Science, The
University of Melbourne, Parkville, VIC 3010, Australia
and yield components [5]. The photosynthetic traits such as
chlorophyll content, chlorophyll fluorescence parameters and
leaf gas exchange could help in assessing the influence of
the drought stress on crop growth and yield, as these traits
are closely correlated with yield related traits [6, 7]. Infrared
thermal imaging of crop canopies also provides an efficient
non-destructive tool in the quantification of plant responses
to water stress in controlled and field conditions [8–11].
Silicon (Si) is the second most abundant element in
the soil and often regarded as an essential plant nutrient
[12] with beneficial effects ameliorating biotic and abiotic
stresses in plants [13–15]. Si is also involved in regulat-
ing several physiological and metabolic processes in plants
under stressed and non-stressed conditions [16]. Supple-
mentation of Si has been adopted as an effective strategy
for alleviating the negative effects of drought stress and
improving the drought tolerance of plants [16–20]. Early
reports have documented the effects of Si on photosynthesis
and chlorophyll fluorescence of plants under drought stress
[21–24]. The investigation on Si mediated alleviation of

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drought stress in plants has largely been restricted to higher
Si accumulating monocots and studies of low Si accumulat-
ing dicots are relatively few [25–27].
It has been demonstrated that Si enhances polyethylene
glycol (PEG) induced drought stress tolerance in lentil seed-
lings [16]. To best of our knowledge, no previous studies
have addressed the role of Si in regulating the photosyn-
thetic efficiency, infrared thermal canopy temperature (IRTc)
and yield components of drought stressed lentil plants.
Therefore, this study was conducted to further explore the
defensive role of Si in lentil under drought stress regarding
regulating photosynthetic mechanism and yield components.
The results from this study may partially help to elucidate
the underlying physiological mechanisms of Si induced
improvement of drought tolerance of the lentil plants.
2 Materials and Methods
2.1 Plant Materials
Lentil genotypes (procured from the Australian Grains
Gene Bank, Victoria) used in this study were selected from
a drought tolerance screening experiment based on infrared
thermal imaging [11]. The genotypes were selected from
three categories, drought tolerant (ILL 6002; G1), moder-
ately drought tolerant (PBA Jumbo 2; G2) and drought sus-
ceptible (ILL 7537; G3). This study was initially conducted
in a controlled growth room (temperature: 23 ± 2 °C; rela-
tive humidity: 45–50%; photoperiod: 12 h; light intensity:
300–325 μmol ­m2 ­s−1 from metal halide illumination lamps
(MH 400 W/640 E40 CLU 1SL/6, Netherlands) of the Uni-
versity of Melbourne, Parkville to record the photosynthetic
and yield related traits [11]. Subsequently, a field experiment
was also undertaken to validate the results from the growth
room experiment.
2.2 Experiment Under Controlled Conditions
(Growth Room)
Studies on photosynthetic efficiency were performed to
understand the behaviour of selected lentil genotypes under
severe drought stress and Si application. The experiment
was established as a completely randomised design with four
treatments in triplicates. The treatments were as follows:
control (C—well watered, 100% FC), severe drought stress
(D—20% FC), severe drought stress with supplementary
addition of Si (DSi), and Si alone (Si). Lentil seeds were
surface sterilised with 30% (v/v) hydrogen peroxide solution
and sown in plastic pots (950 ml) containing 700 g potting
mix. Source of Si was Sodium metasilicate ­(Na2SiO3) [16]
and 2 mM of N ­ a2SiO3 (500 ml ­kg−1 potting mix) was added
to the pots prior to seed sowing. The molarity of ­Na2SiO3
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was calculated based on the volume of potting mix and the
pH of the solution was adjusted to 7.5 using 0.1 N hydro-
chloric acid. Sodium sulphate ­(Na2SO4; 2 mM) was added
to the control pots to balance the sodium levels and the basi-
fication due to Si. Nitrosol (Amsgrow) is used to fertilise the
plants during the vegetative phase to maintain normal plant
growth. Plants were subjected to drought stress at the onset
of flowering period at respective field capacities. All the
measurements for photosynthetic pigments, chlorophyll fluo-
rescence, gas exchange parameters and canopy temperature
were carried out after 28 days from the initiation of drought
stress. All the plants were harvested at maturity and plant
height, above ground biomass, and yield related traits were
measured after drying at 40 °C for 72 h.
2.3 Experiment in Field Conditions
A field experiment was conducted from May—December
2018 at the Dookie campus farm, The University of Mel-
bourne (36.378S, 145.708E, 185 m above sea level in Vic-
toria, Australia, using a total area of 308 ­m2 (14 m x 22 m).
The rainfall, relative humidity, and air temperature data dur-
ing the entire cropping season (May—December 2018) were
recorded from an automated meteorological station located
at the Dookie weather station (Adcon Telemetry one, New
South Wales, Australia) (Table 1). The experiment was
laid out in randomized block design with three genotypes
and three replicates using a split plot arrangement in three
blocks. The soil at the experimental site was red brown clay
with a pH 6.5. Sodium meta silicate (­ Na2SiO3; 106 mg/Kg
soil) was applied to the treatment plots, one week before
seed sowing. Sodium sulfate (71 mg/Kg) was added to the
control plots at the same time to minimize the medium basi-
fication as well as sodium effects originating from N ­ a2SiO3
addition. The volume of Si applied was calculated, based on
already available Si content in soil and the dry bulk density
of soil. The added Si was mixed well with soil up to 15 cm
depth in each corresponding treatment plot soil analysis was
carried out before and after Si application. Three soil sam-
ples were randomly taken, to a depth of 0–6 cm, from each
plot. The soil samples were homogenized, and two compos-
ite samples were analysed for physical and chemical proper-
ties of soil at the Nutrient Advantage company, Werribee,
Australia. Fertilizers were incorporated at the rate of 20 kg
urea ­ha−1 and 18 kg superphosphate h­ a−1 before sowing.
The three genotypes were randomised within each plot and
planted in five rows spaced, which were 50 cm apart. Plants
were spaced at 25 cm from each other in each row. Buffer
zones (0.5 m) were established (2 m buffer zone in between
blocks and 0.5-m buffer zone between the plots) to minimize
potential Si contamination via lateral movements.
Inoculated seeds (Group F® Rhizobium legumino-
sarum) were hand sown at a sowing depth of 2.5 cm. The
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Table 1  Weather parameters at the Dookie farm during the crop season (May—December 2018) and soil properties before and after silicon addi-
tion
Month May June July August September October November December

Total rainfall (mm) 23.11 20.41 13.21 34.16 4.81 19.61 51.22 71.62
Average temperature 11.71 8.32 8.11 7.81 9.92 15.63 18.11 23.61
(oC)
Relative humidity 73.41 83.52 80.01 79.72 70.31 59.13 58.23 51.64
(%)
Soil parameters pH Electrical Si (mg/g) Nitrate-Nitrogen Phosphorous Potassium Organic Carbon Sodium (mg/
conductivity (mg/g) (mg/g) (mg/g) (%) Kg)
(dS/m)
Before Si addition 6.50 1.10 120.00 55.00 48.00 2.20 2.20 0.11
After Si addition 7.70 3.60 212.00 90.00 64.00 2.30 2.30 0.12

plots were irrigated after sowing to guarantee seed ger-
mination and consistent emergence of seedlings. Subse-
quently, plots were irrigated once per week (except for
those weeks, when there was a rainfall event) until the
onset of imposed drought stress for the drought stressed
plots (no rainfall events occurred during this time), while
non-stressed control plots were maintained with regular
irrigations until physiological maturity of the crop.
The treatments were as control (C-well irrigated, no
drought stress), drought stress (D), drought stress with
supplementary Si (DSi), and Si alone (Si). Plants were
subjected to drought stress (Mid-September) by withhold-
ing irrigation at flowering stage (R2 stage). Estimation
of photosynthetic pigments, chlorophyll fluorescence, gas
exchange parameters and canopy temperature measure-
ments were carried out on the ­2 8 th day, from the onset
of drought stress treatments. Volumetric soil moisture
content in each plot (taken from an average of 5 spots
from one plot) was determined with a soil moisture sensor
(Theta probe, ML2, USA), throughout the experimental
period, on a fortnightly basis. The plots were kept free of
weeds by hand weeding. Plants from all treatments were
harvested in December when more than 90% of the pods
had turned golden-brown. Harvesting was done in all the
plots from an area of 1 m2 (2 × 0.5 m2 quadrats, 4 rows)
after removing the plot border rows on either end of the
plot to avoid edge effect. Plant height (cm), from the base
of the plant to the top of the main shoot, was measured
at maturity. Above ground biomass (g) and yield related
components such as number of pods and seeds, pod weight
and seed yield were measured after drying harvested plants
at 40 °C for 72 h. Harvest index (HI) was calculated as a
ratio of seed yield to the above-ground dry matter. Seed
yield per plot was converted to kilogram per hectare (Kg
­ha−1) for statistical analysis.
2.4 Estimation of Photosynthetic Pigments
The concentration of photosynthetic pigments (chlorophyll
a, chlorophyll b, total carotenoids) were measured from lentil
leaves grown in both the growth chamber and the field [28].
For this purpose, leaf sample (1 g) were ground in 5 mL of
80% acetone using a mortar and pestle. The homogenate was
centrifuged at 6000 rpm for 10 min. The supernatant was
decanted into another centrifuge tube and diluted with 80%
acetone to adjust the final volume to 10 mL. The absorb-
ances at 470 nm, 646.8 nm and 663.2 nm were measured
using a spectrophotometer (M501, Campspec Ltd, Cam-
bridge, UK) with 80% acetone acting as a blank. The con-
centration of pigments in mg m ­ L−1 was calculated using the
following equations [29]:
Chlorophyll a(Ca) = 12.25A663.2 − 2.79A646.8 (1)
Chlorophyll b(Cb) = 21.50A646.8 − 5.10A663.2 (2)
Total carotenoid = (1000A470 − 1.82Ca − 85.02Cb)∕198
(3)
where A663.2 is the absorbance at 663.2 nm, A646.8 is
absorbance at 646.8 nm and A470 is the absorbance at
470 nm.
2.5 Chlorophyll Fluorescence
Chlorophyll fluorescence was measured on the youngest
fully expanded leaf of lentil grown in both the growth cham-
ber and the field, using a MINI-PAM II portable chlorophyll
fluorometer (MINI-PAM II analyzer, Heinz Walz GmbH,
Effeltrich, Germany) [30]. The analyzer was connected to

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a leaf-clip holder (2030-B), which uses a 60° fibre optic Thermal and visible images were co-registered to differ-
attachment to simultaneously record photosynthetically entiate between the canopy and the background by exclud-
active radiation (PAR) and temperature. The measurements ing the background, soil and pot in the growth chamber
were taken under steady-state light source at photosynthetic and soil in the field. The thermal images were processed to
photon flux density of ~ 150 µmol·m−2·s−1, which is adequate obtain IRTc and crop water stress index (CWSI) according
to excite the phytochrome within PAR (400–700 nm). Sam- to the equations given in [11].
pling was performed using MODE 1, which is the standard
mode of measurement for yield parameters (ΔF/Fm). The
AUTO-ZERO function mode (MODE 2) was used after 2.8 Statistical Analysis
every six samplings to clear any false signal. The quantum
yield of photosystem II was measured using the equation All the experiments were performed in triplicates. Statisti-
[30]: cal analysis of the data was done using analysis of vari-
( � �
ance (ANOVA), followed by a Tukey pairwise comparison
(4)
)
ΦPSII = Fm − Ft ∕Fm test for mean comparison between genotypes and treat-
ments using Minitab®v17 (Minitab Inc., Pennsylvania,
where Fm’ is the maximum fluorescence when actinic light
USA). A multivariate data analysis, based on principal
is applied. Ft is the level of fluorescence immediately before
components analysis (PCA), cluster analysis and correla-
the saturating light flash. The proportion of light that entered
tion matrix, was carried out for the data obtained from
Photosystem II is quantified from this equation. The effec-
the growth chamber and the field, by using a customized
tive quantum yield of PSII-Y(II) was the main parameter
code written in MATLAB® R2019a. The PCA was used
used in this study.
to find relationships between all the studied parameters
collected from the growth chamber and field experiments.
2.6 Leaf Gas Exchange Parameters The cluster analysis was used to classify the samples based
on different stress treatments, and the correlation matrix
Leaf gas exchange parameters such as net photosynthesis was generated to assess the statistically significant corre-
(Pn), stomatal conductance (gs); intercellular ­CO2 concen- lations (P ≤ 0.05) between the studied parameters in both
tration (Ci) and transpiration rate (Tr) were measured on the experiments. The factor loadings of PCA are given in
the youngest fully expanded leaves of lentil grown in the Table 2.
growth chamber and the field using a portable photosynthe-
sis system (Li-6400, Li-COR, Lincoln, NE, USA) equipped
with a 6 c­ m2 chamber. The gas exchange analyser quantifies
photosynthesis and transpiration by measuring C
­ O2 and ­H2O
flow with an infrared gas analyser (IRGA). Measurements
were done considering 400 μmol ­CO2 ­mol−1 air at photo-
Table 2  Factor loadings of the two principal components for each
synthetic photon flux density of ~ 150 µmol·m−2 ·s−1 and the parameter from controlled and field conditions
relative humidity was maintained at 45–50%. Leaf area was
Controlled conditions Field conditions
measured by capturing images using an iphone 6S mobile
(Apple, Australia) and analysing with MATLAB (Math- Parameter PC1 PC2 PC1 PC2
Works® R2018b) using a customized computer vision code Fv/Fm 0.31 0.18 0.33 0.21
and photosynthesis rate was calculated per ­cm2 leaf area. Chl a 0.30 0.3 0.33 -0.07
Chl b 0.32 0.07 0.33 -0.08
2.7 Infrared Thermal Canopy Temperature (IRTc) Carotenoids 0.30 -0.05 0.33 0.09
and Crop Water Stress Index (CWSI) Pn 0.32 -0.21 0.33 0.07
gs 0.29 0.14 0.34 0.01
Infrared thermal images (IRT) of plants from the growth Tr -0.20 0.79 -0.17 -0.60
chamber and the field were captured using FLIR T-series Ci 0.30 0.09 0.20 -0.30
(Model B360) (FLIR Systems, Portland USA) infrared IRTc -0.30 -0.28 -0.1 0.63
camera. Thermal images were taken from the canopy of CWSI -0.30 0.01 -0.32 -0.04
the genotypes in the growth chamber and in each plot in Seed yield 0.30 -0.28 0.31 0.25
the field. The images were processed and analysed using
The abbreviations used in Table are Chl a-chlorophyll a, Chl b-chlo-
proprietary MATLAB code (MATLAB R2018b, Math-
rophyll b, Pn—photosynthetic rate, gs—stomatal conductance, Tr—
works Inc., Natick, MA, USA) and the Image Analysis transpiration rate, Ci—internal C­O2 concentration, IRTc-infrared
Toolbox to estimate IRT canopy temperatures (IRTc). thermal canopy temperature and CWSI-crop water stress index

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3 Results
3.1 Weather Conditions and Soil Parameters
of the Field Trial
The average air temperature (Ta) during the entire experi-
ment ranged from 7.81 – 23.61 °C, which was within the
ideal range (Ta < 32 °C) for lentil growth. However, the
amount of total rainfall received throughout the growing sea-
son was much lower than the 300–500 mm rainfall required
for the optimal growth of the crop [31] indicating a preva-
lence of moderate to severe drought stress during this crop-
ping season. Therefore, an irrigation scheduling was main-
tained to avoid any additional stress conditions for drought
stress treatment plots, other than the imposed drought stress
period and to maintain steady moisture in non-stressed con-
trol plots. As displayed in the Table 1, the relative humidity
(RH) was moderately high i.e., 70—84% from the seedling
emergence stage (VE) to the flowering stage (R2); however,
during the imposed drought stress stage, RH was optimum
(nearly 65%) in the field.
Field soil texture was clay loam with a reddish-brown
colour and a pH of 6.5. Total soil potassium (K), sodium
(Na) and organic carbon levels did not show any signifi-
cant differences before and after Si addition (Table 1). Plant
available Si (PAS/ (­ CaCl2—extractable Si) levels in the soil
depends on soil type and usually clayey soils contain 200 to
300 g Si ­kg−1 [32], however, the Si concentration in the soil
from the experimental site was only 120 mg ­kg−1 of soil.
PAS levels in the soil increased significantly after Si addi-
tion, with an increase in soil pH to 7.7. This was within the
ideal range (6–8) for lentil production [31]. Soil moisture
content increased significantly in Si applied plots compared
with the control plots and even under drought stress condi-
tions (Fig. 1), which revealed that Si can improve the water-
holding capacity of the soil [33].
Fig. 1  Average volumetric soil
moisture content (­ m3.m−3) from
treatment plots during the crop-
ping season, May—December
2018. The abbreviations used in
this Figure are C-control, Si-Si
alone, D- drought stress and
DSi-drought stress supple-
mented with Si (DSi) treat-
ments.
3.2 Fv/Fm Ratio and Photosynthetic Pigments
Under both controlled and field conditions, drought stress
significantly decreased photosynthetic yield (as assessed by
Fv/Fm ratio) and photosynthetic pigments (chlorophyll a,
chlorophyll b and total carotenoids) of all the studied geno-
types, compared with the non—stressed genotypes (Figs. 2
and 3). In contrast, the Fv/Fm ratio and photosynthetic pig-
ments content of all the drought stressed genotypes were
increased significantly in response to Si supplementation.
However, the increase in photosynthetic yield, chlorophyll
a and chlorophyll b contents of the non-stressed genotypes
was non-significant with Si application compared with the
control plants. A significant increase in levels of total carote-
noids was also noticed in non-stressed genotypes in response
to Si, compared with the control. Furthermore, there were
no significant differences for PS II function and levels of
photosynthetic pigments among the genotypes, even though
they belonged to different drought tolerance categories.
3.3 Leaf Gas Exchange Parameters
Drought stress led to a considerable decrease in the values of
gas exchange parameters such as Pn, Gs, Tr and Ci in all the
genotypes under drought stress, compared with the control
(controlled and field conditions) (Tables 3 and 4). The posi-
tive effect of Si on gas exchange parameters was significant
in all the drought stressed lentil genotypes in response to Si,
compared with their non-Si treated genotypes grown under
drought stress. In Si treated drought stressed genotypes, val-
ues of Pn increased by 1.2–2.3 folds, Gs by 0.5–1.0 folds,
Tr by 1.5 folds and Ci by 1.1–1.6 folds compared with the
drought stressed genotypes. The addition of Si under drought
stress changed mean values of Ci in drought susceptible gen-
otypes which were statistically similar to moderately drought
tolerant and tolerant genotypes.
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Fig. 2  (A) Fv/Fm ratio, (B) chlorophyll a (C) chlorophyll b and (D)
total carotenoids in lentil genotypes under different drought stress
treatments in controlled conditions. Abbreviations used in this Fig-
ure are, C- control, Si- Si alone, D- drought stress and DSi- drought
3.4 Canopy Temperature (Tc) and Crop Water Stress
Index (CWSI)
Canopy temperature (IRTc) of drought stressed lentil geno-
types increased significantly (27–28 °C in controlled and
34–36 °C field conditions) compared with their respective
controls (16–17 °C in controlled and 29–31 °C in field con-
ditions) (Figs. 4 and 5). Supplementation of Si to drought
stressed plants resulted a significant reduction in IRTc and
were in the range of 17–19 °C and 16–19 °C under con-
trolled and field conditions, respectively. However, IRTc was
in the range of 13–14 °C in controlled and 23–24 °C in field
conditions, with supplementation of Si in the non-stressed
genotypes.
3.5 Aboveground Biomass and Yield Traits
Plant height, above ground biomass and the yield related
traits (pod number, pod weight, seed number and seed yield)
increased significantly in genotypes under drought stress and
Si supplementation, compared with drought stress with no
added Si, under controlled and field conditions (Tables 5
and 6). However, Si alone treatment revealed non-significant
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stress supplemented with Si. Genotypes are, G1- ILL 6002, G2- PBA
Jumbo 2, G3- ILL 7537. Mean values with error bars represent the
standard deviation and different letters represent statistical signifi-
cances within the genotypes (Tukey test; p ≤ 0.05)
differences for all the above-mentioned traits among the dif-
ferent genotypes compared with their respective controls.
Seed yield in the Si applied drought stress treatment, under
controlled condition, increased 1.4 folds in drought toler-
ant genotypes, 1.7 folds in moderately tolerant genotypes
and 6 folds in susceptible genotypes, as compared with the
same genotypes grown in drought stress treatment. In the
field condition, the drought stressed genotypes showed an
increase by 1.5, 1.7 and 2.9 folds for grain yield respec-
tively in the drought tolerant, moderately drought tolerant
and drought susceptible genotypes in response to Si supple-
mentation compared with their respective drought stressed
genotypes without Si treatment.
3.6 Correlations Between Growth Room and Field
Experiments Using Multivariate Data Analysis
The results from multivariate data analysis from all the four
treatments are shown in Fig. 6a (controlled experiment) and
6b (field experiment). The PCA biplot obtained from the
studied lentil genotypes under control (C), Si alone (Si),
drought stress (D) and drought stress supplemented with
Si (DSi) treatments including the photosynthetic variables,
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Fig. 3  (A) Fv/Fm ratio, (B) chlorophyll a (C) chlorophyll b and (D) supplemented with Si. Genotypes are, G1- ILL 6002, G2- PBA
total carotenoids in lentil genotypes under different drought stress Jumbo 2, G3- ILL 7537. Mean values with error bars represent the
treatments in field conditions. Abbreviations used in this Figure are, standard deviation and different letters represent statistical signifi-
C- control, Si- Si alone, D- drought stress and DSi- drought stress cances within the genotypes (Tukey test; p ≤ 0.05)

Table 3  Leaf gas exchange parameters in lentil genotypes under different drought stress treatments in controlled conditions

Genotype Treatment Pn gs Tr Ci
(µmol ­CO2 ­m−2 s −1) (mol ­H2O ­m−2 ­s−1) (mmol ­H2O ­m−2 s −1) (µmol ­CO2 ­mol−1)

G1 C 7.85 ± 0.23a 1.20 ± 0.31a 2.08 ± 0.13c 356.32 ± 3.52b

Si 7.89 ± 0.22a 1.28 ± 0.81a 2.14 ± 0.65c 320.36 ± 3.14a
D 4.59 ± 0.12c 0.50 ± 0.104c 4.78 ± 0.22b 205.36 ± 2.52d
DSi 5.67 ± 0.16b 0.10 ± 0.01b 7.34 ± 0.12a 315.01 ± 2.81c
G2 C 7.84 ± 0.26a 1.32 ± 0.12a 2.16 ± 0.34c 352.69 ± 2.75b
Si 7.70 ± 0.23a 1.34 ± 0.63a 2.65 ± 0.17c 301.37 ± 3.83a
D 3.65 ± 0.13c 0.96 ± 0.113a 4.52 ± 0.55b 198.24 ± 2.82c
DSi 4.88 ± 0.10b 0.30 ± 1.32b 7.21 ± 0.36a 309.33 ± 2.94d
G3 C 7.62 ± 0.24a 1.30 ± 0.44a 2.66 ± 0.41c 352.87 ± 3.92a
Si 7.83 ± 0.23a 1.28 ± 0.11a 2.92 ± 0.12c 354.24 ± 3.02b
D 2.74 ± 0.19c 1.00 ± 0.20a 6.12 ± 0.43b 121.32 ± 3.81d
DSi 4.26 ± 0.15b 0.40 ± 0.14b 8.91 ± 0.13a 289.97 ± 1.74c

Abbreviations used in this table for studied parameters (Pn—photosynthetic rate, gs—stomatal conductance Tr—transpiration rate, Ci—internal
­CO2 concentration), genotypes (G1—ILL 6002, G2—PBA Jumbo 2, G3—ILL 7537) and treatments (C—control, Si—Si alone, D—drought
stress, and DSi—drought stress supplemented with Si). Mean values are provided with standard deviation and different letters represent statisti-
cal significances within the genotypes (Tukey test; p ≤ 0.05)

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Table 4  Leaf gas exchange parameters in lentil genotypes under different drought stress treatments in field conditions
Genotype Treatment Pn gs Tr Ci
(µmol ­CO2 ­m−2 s −1) (mol ­H2O ­m−2 ­s−1) (mmol ­H2O ­m−2 s −1) (µmol ­CO2 ­mol−1)

G1 C 10.35 ± 0.38b 1.50 ± 0.35a 3.31 ± 0.34d 378.36 ± 3.35a

Si 12.35 ± 0.66a 1.65 ± 0.33a 5.35 ± 0.25c 386.89 ± 3.65a
D 06.35 ± 0.33c 0.75 ± 0.23a 7.36 ± 0.24b 305.36 ± 5.36b
DSi 8.98 ± 0.67ab 1.25 ± 0.55a 9.35 ± 0.28a 391.36 ± 3.37a
G2 C 9.68 ± 1.23b 1.33 ± 0.25a 3.16 ± 0.98d 376.32 ± 2.35b
Si 12.25 ± 1.12a 1.50 ± 0.54a 5.16 ± 0.35c 387.22 ± 0.68b
D 6.01 ± 0.25c 0.40 ± 0.15b 7.05 ± 0.65b 388.36 ± 5.32c
DSi 8.36 ± 0.98b 0.22 ± 0.09b 9.23 ± 0.55a 301.72 ± 4.96a
G3 C 11.23 ± 1.02a 1.43 ± 0.02a 3.23 ± 0.87d 368.38 ± 2.37b
Si 12.36 ± 1.01a 1.63 ± 0.34a 6.11 ± 0.52c 313.98 ± 3.54a
D 3.25 ± 0.85c 0.51 ± 0.05b 8.07 ± 0.56b 389.36 ± 2.35c
DSi 7.65 ± 0.02b 0.20 ± 0.35b 9.23 ± 0.99a 352.03 ± 4.92a

Abbreviations used in this table for studied parameters (Pn—photosynthetic rate, gs—stomatal conductance Tr—transpiration rate, Ci—internal
­CO2 concentration), genotypes (G1—ILL 6002, G2—PBA Jumbo 2, G3—ILL 7537) and treatments (C—control, Si—Si alone, D—drought
stress, and DSi- drought stress supplemented with Si). Mean values are provided with standard deviation and different letters represent statistical
significances within the genotypes (Tukey test; p ≤ 0.05)

canopy measurements and seed yield explained a total of
94.5% (PC1-85.9%; PC2-8.60%) of variability in the data
obtained from controlled conditions and 96.3% ( PC1-
80.19% and PC 2–16.54%) of variability obtained from field
conditions. Four distinct groups were identified in both PCA
biplots, which corresponded to the different treatments, such
as control, Si alone, drought and drought supplemented with
Si. The cluster analysis showed two main clusters in both
the cluster diagrams obtained from the controlled and field
conditions (Fig. 7). All the drought stressed genotypes (D)
were clustered together in one small cluster and the other
large cluster included genotypes from control (C) and Si
supplemented drought stress (DSi) and Si alone (Si) treat-
ments. Both correlation matrix diagrams showed significant
positive correlations among and between chlorophyll pig-
ments, Fv/Fm ratio, Pn, gs, Ci and seed yield (Fig. 8).
4 Discussion
4.1 Silicon Maintains Fv/Fm Ratio
and Photosynthetic Pigments in Drought
Stressed Lentil
Chlorophyll and carotenoid pigments are the major chlo-
roplast components responsible for harvesting light and
the oxidation -reduction reactions in plants [34]. Photo-
synthetic yield is a relative measure of the rate of linear

Fig. 4  (A) Infrared thermal canopy temperature (IRTc) and (B) crop drought stress supplemented with Si. Genotypes are, G1- ILL 6002,
water stress index (CWSI) in lentil genotypes under different drought G2- PBA Jumbo 2, G3- ILL 7537. Mean values with error bars rep-
stress treatments, grown in controlled conditions. Abbreviations used resent the standard deviation and different letters represent statistical
in this Figure are, C- control, Si- Si alone, D- drought stress and DSi- significances within the genotypes (Tukey test; p ≤ 0.05)

13
Silicon

Fig. 5  (A) Infrared thermal canopy temperature (IRTc) and (B) crop drought stress supplemented with Si. Genotypes are, G1- ILL 6002,
water stress index (CWSI) in lentil genotypes under different drought G2- PBA Jumbo 2, G3- ILL 7537. Mean values with error bars rep-
stress treatments, grown in field conditions. Abbreviations used in resent the standard deviation and different letters represent statistical
this Figure are, C- control, Si- Si alone, D- drought stress and DSi- significances within the genotypes (Tukey test; p ≤ 0.05)

electron transport in the reaction centres of PSII and can be
used to approximate the extent of drought stress damage to
chloroplasts [35]. It can be speculated that PS II yield is a
non-stomatal limitation to photosynthesis. It is expected for
photosynthetic yield and photosynthetic pigment levels to
decrease under drought stress because of reactive oxygen
species (ROS) generation, which damages or inactivates the
PSII reaction centres in chloroplast [36, 37]. The observed
effect of Si in increasing photosynthetic yield and photosyn-
thetic pigment levels under drought stress might be due to
the balanced antioxidant activity in drought stressed lentil
genotypes [16] that prevented damage to PSII reaction cen-
tres caused by ROS. This may also explain why Si had no
effect on non-stressed genotypes. The increased carotenoid
levels, even in non-stressed genotypes, in response to Si
clearly indicates its photoprotective role in quenching tri-
plet state chlorophyll molecules and scavenging ROS formed
within the chloroplast [38]. Moreover, these results could
also be explained based on Kaufman et al.’s [39] findings
in sugarcane cultivars, which suggest that Si deposited in
epidermal cells as silica, could enhance light-use efficiency
by helping the light transmission to the photosynthetically
active mesophyll tissues in leaves, as revealed by scanning
electron microscopy (SEM), X-ray analysis and light micros-
copy studies. The authors suggested that silica cells in sug-
arcane cultivars act like windows in the epidermal system,
allowing more light to be transmitted to photosynthetic tis-
sues than other adjacent epidermal cells.
Wang et al. [40] added strong evidence to support the
role of Si in alleviating damage to the photosystem caused

Table 5  Plant height, above ground biomass, pod number, pod weight, seed number and seed weight of lentil genotypes in controlled conditions

Genotype Treatment Plant height Aboveground Pod number/ Pod weight Seed number/ Seed yield/plant Seed yield/pot
(cm) biomass/plant plant /plant (g) plant (g) (Kg/ha)
(g)

G1 C 65.32 ± 1.36a 10.62 ± 0.31a 167.89 ± 0.23b 4.95 ± 0.01a 230.12 ± 0.09b 4.00 ± 0.56b 571.42 ± 0.65b
Si 63.32 ± 1.02a 10.30 ± 0.53a 234.23 ± 0.44a 5.98 ± 0.48a 356.87 ± 0.23a 5.13 ± 0.55a 732.85 ± 0.35a
D 50.32 ± 0.69b 7.3 ± 0.33c 65.28 ± 0.57c 2.05 ± 0.01b 116.22 ± 0.45c 1.66 ± 0.52d 237.14 ± 0.97d
DSi 59.37 ± 2.30ab 9.9 ± 0.62b 109.02 ± 0.25b 3.05 ± 0.75ab 204.18 ± 0.86ab 2.35 ± 0.11c 335.71 ± 0.88c
G2 C 60.36 ± 1.22a 8.8 ± 0.81a 155.02 ± 0.24b 5.25 ± 0.04a 287.36 ± 0.05b 4.57 ± 0.41a 652.85 ± 0.78b
Si 65.36 ± 1.28a 9.5 ± 0.81a 222.03 ± 0.88a 6.52 ± 0.44a 424.36 ± 0.65a 4.98 ± 0.44a 711.42 ± 0.97a
D 44.36 ± 2.36c 5.6 ± 0.55c 45.39 ± 0.65d 2.09 ± 0.07c 105.98 ± 0.45c 1.48 ± 0.01b 211.42 ± 0.76d
DSi 53.36 ± 0.67b 7.6 ± 0.56b 102.30 ± 0.77c 3.14 ± 0.56b 226.37 ± 0.45ab 2.53 ± 0.01c 361.42 ± 0.82c
G3 C 38.69 ± 1.33a 8.97 ± 0.69a 145.01 ± 0.25b 3.26 ± 0.11b 267.91 ± 0.54b 2.96 ± 0.33b 422.85 ± 0.77b
Si 39.25 ± 1.23a 9.68 ± 0.75a 209.56 ± 0.11a 5.34 ± 0.50a 410.37 ± 0.65a 4.81 ± 0.52a 687.14 ± 0.73a
D 19.36 ± 02.32c 2.65 ± 0.63c 26.01 ± 0.52d 0.35 ± 0.01d 49.32 ± 0.78d 0.17 ± 0.02d 242.28 ± 0.94d
DSi 28.63 ± 0.38b 5.64 ± 0.82b 93.32 ± 0.25c 1.62 ± 0.78c 165.07 ± 0.65c 1.04 ± 0.62c 571.42 ± 0.73c

Genotypes are G1- ILL 6002, G2- PBA Jumbo 2 and G3- ILL 7537. Abbreviations used in this table are, C—control, Si—Si alone D—drought
stress and DSi—drought stress supplemented with Si. Mean values are provided with standard deviation and different letters represent statistical
significances within the genotypes (Tukey test; p ≤ 0.05)

13
 Silicon

Table 6  Plant height, above ground biomass, pod number, pod weight, seed number and seed weight of lentil genotypes under field conditions
Genotype Treatment Plant height Aboveground Pod number/ Pod weight/ Seed number/ Seed yield/ Seed yield/plot
(cm) biomass/plant plant plant (g) plant plant (g) (Kg/ha)
(g)

G1 C 33.23 ± 1.32a 5.79 ± 0.33a 39.75 ± 1.23a 2.96 ± 0.01a 301.25 ± 1.02b 2.65 ± 0.12a 397.51 ± 2.22a
Si 33.32 ± 1.64a 6.58 ± 0.04a 43.35 ± 2.36a 3.11 ± 0.23a 475.32 ± 2.11a 2.89 ± 0.02a 433.25 ± 3.33a
D 25.62 ± 0.34b 3.27 ± 0.01c 19.25 ± 2.24c 1.65 ± 0.12b 156.25 ± 3.21c 1.35 ± 0.13b 202.95 ± 2.36c
DSi 32.75 ± 0.55a 4.63 ± 0.23ab 26.71 ± 0.27b 2.14 ± 0.87ab 335.65 ± 2.33b 1.78 ± 0.01b 267.02 ± 1.23b
G2 C 34.97 ± 0.31a 5.87 ± 0.02a 35.25 ± 1.02b 2.68 ± 0.27b 324.56 ± 2.37b 2.35 ± 0.34b 352.52 ± 3.21b
Si 33.57 ± 0.79a 6.65 ± 0.23a 44.72 ± 1.25a 3.45 ± 0.22a 432.32 ± 2.87a 2.98 ± 0.01b 447.33 ± 3.26a
D 23.62 ± 0.40b 2.15 ± 0.01c 15.33 ± 0.97d 1.65 ± 0.88b 112.86 ± 3.65d 1.02 ± 0.07b 153.39 ± 2.23d
DSi 30.23 ± 0.85a 4.67 ± 0.21ab 23.27 ± 1.77c 2.12 ± 0.32ab 220.36 ± 2.93c 1.78 ± 0.21ab 267.32 ± 2.35c
G3 C 31.96 ± 0.74a 5.87 ± 0.03a 41.25 ± 0.88a 3.12 ± 0.23a 254.78 ± 4.23b 2.75 ± 0.16a 412.51 ± 2.18a
Si 32.32 ± 0.73a 6.69 ± 0.98a 45.15 ± 1.85a 3.44 ± 0.27a 389.36 ± 2.37a 3.01 ± 0.66a 451.53 ± 1.36a
D 20.77 ± 1.72b 1.02 ± 0.05c 13.33 ± 0.32c 0.99 ± 0.11b 57.66 ± 4.83d 0.22 ± 0.75c 33.36 ± 1.32c
DSi 33.25 ± 0.52a 2.35 ± 0.44b 29.75 ± 0.65b 1.94 ± 0.23c 148.36 ± 1.32c 0.65 ± 0.33b 97.53 ± 3.23b

Genotypes are G1- ILL 6002, G2- PBA Jumbo 2 and G3- ILL 7537. Abbreviations used in this table are, C—control, Si—Si alone D—drought
stress and DSi—drought stress supplemented with Si. Mean values are provided with standard deviation and different letters represent statistical
significances within the genotypes (Tukey test; p ≤ 0.05)

by drought stress by altering the thylakoid membrane
protein components in rice seedlings. The authors found
that Si upregulated chlorophyll a-b binding proteins, PS II
reaction centre core proteins and ATP synthase subunits,
which are responsible for the light energy transformation
reactions taking place in the thylakoid membrane of the
chloroplasts. These upregulations improved light harvest-
ing efficiency, the stability of PS I and PS II reaction
centres, electron transport, PS II core antenna content,
and ATP synthesis. Taken together, the current study
clearly indicates the role of Si in protecting PSII reaction
centres in thylakoids, by maintaining ROS homeostasis to
improve photosynthetic performance under drought stress
conditions, as evidenced in several other crops including
Sorghum [41, 42], Soybean [27], Rice [1], Pea [43] and
Wheat [44, 45].

Fig. 6  PCA biplot (A) from controlled and (B) field conditions of len-
til genotypes under different drought stress treatments. Abbreviations
used in this Figure are, C- control, Si- Si alone, D- drought stress and
DSi- drought stress supplemented with Si, Chl a-chlorophyll a, Chl
b-chlorophyll b, Pn—photosynthetic rate, gs—stomatal conductance,
Tr—transpiration rate, Ci—internal ­CO2 concentration, IRTc-infra-
red thermal canopy temperature and CWSI-crop water stress index;
Genotypes are, G1- ILL 6002, G2- PBA Jumbo 2, G3- ILL 7537.
(p ≤ 0.05)

13
Silicon
Fig. 7  Cluster diagram from (A) controlled and (B) field condi-
tions of lentil genotypes under different drought stress treatments.
The abbreviations used in this Figure are C-control, Si–Si alone, D-
4.2 Si Regulates Leaf Gas Exchange Parameters
Under Drought Stress in Lentil Genotypes.
Drought induced reduction in photosynthesis has been pre-
viously studied in lentil [5]. This reduction is usually clas-
sified according to whether it is related or not related to
stomatal limitations [46]. These stomatal limitations result
from the resistance to C­ O2 diffusion into the intercellular
leaf spaces via stomatal closure [47], whereas, non—sto-
matal limitations include all other factors, such as diffusive
(reduced mesophyll conductance) and metabolic (photo-
chemical and enzymatic reactions) processes that could
Fig. 8  The correlation matrix from (A) controlled and (B) field con-
ditions of lentil genotypes drought stress supplemented with Si (DSi)
treatments. The abbreviations used in Figure are Chl a-chlorophyll a,
Chl b-chlorophyll b, Pn—photosynthetic rate, gs—stomatal conduct-
drought stress and DSi-drought stress supplemented with Si (DSi)
treatments. Genotypes are G1—ILL 6002, G2—PBA Jumbo 2, G3—
ILL 7537 and p value ≤ 0.05
decrease photosynthesis or cause chloroplast damage [48].
Si has been proven to alter the non-stomatal limitations in
rice plants by preventing the destruction of photocentric
pigments, maintaining chloroplasts, reducing deterioration
of cell membranes and increasing leaf area, which harvests
more light for photosynthesis [49]. The enhancing effect
of Si on Pn in this study could be attributed mainly to the
non-stomatal limitations observed in this study including
(i) increased chlorophyll pigments (ii) Fv/Fm ratio (Figs. 2
and 3) and (iii) increased above ground biomass (Tables 5
and 6) under Si supplementation of drought stressed lentil
genotypes.
ance, Tr—transpiration rate, Ci—internal C
­ O2 concentration, IRTc-
infrared thermal canopy temperature and CWSI-crop water stress
index
13

The regulation of stomatal movement plays an important
role in controlling gas exchange and balancing water require-
ments, as an efficient adaptive as well as tolerance strat-
egy for plant growth under drought stress conditions [50].
Several studies have shown that stomatal closure reduces
the water loss and simultaneously limits ambient ­CO2 dif-
fusion to the site of carboxylation, which is usually consid-
ered the main reason for the decline in photosynthetic rate
under drought [51]. Supplementation of drought stressed
lentil genotypes with Si increased stomatal conductance
(gs) of leaves, leading to improved influx of ­CO2 to the cel-
lular spaces and resulted in higher Tr values under drought
stress. However, an optimum C ­ O2 concentration in the cel-
lular spaces ensures regular carbon fixation, which in turn
prevents photo-inhibition under drought stress. These results
show that Si influenced the stomatal openings in drought
stressed lentil plants by altering stomatal limitations, which
corroborate with the findings of Gao et al. [52], where Si
application significantly influenced the stomatal open-
ings in maize under drought stress as revealed by scanning
electron and light microscopical studies. As the stomatal
movements are the result of an integrated modulation of ion
fluxes in guard cells [53], further studies should be consid-
ered to elucidate the role of Si in stimulating ion channels
and transporters of guard cells under drought stress. Even
though there was higher water loss through transpiration in
Si treated plants under drought stress conditions, increased
relative water content implied an enhanced water uptake
capacity of the roots. In addition, it can be speculated that,
in the present study, the effect of Si on transpiration loss of
water is subordinate to its effect on growth and improvement
of plants as evidenced by the increased aboveground bio-
mass and yield traits. Taken together, the results suggest that
Si supplementation could improve the water use efficiency
of lentil under drought stress and this effect could contrib-
ute to the alleviation of photosynthetic damage. Moreover,
these results also highlight that Si mitigated drought stress-
induced inhibition of photosynthesis by altering both sto-
matal and non—stomatal mechanisms, which is consistent
with the former studies conducted on other drought stressed
plants including Cucumber[15], Sorghum[41], Wheat[22],
Soybean[27] and Rice[1].
These observations of increased gs and Tr contradicted
some of the previous reports in rice, which showed that Si
led to a decrease in gs and Tr via the formation of a cuticle—
silica double layer, maintaining a high leaf water potential
[54]. However, the finding of this study supports the obser-
vations in rice and in wheat under drought stress, where Si
enhanced both stomatal conductance and transpiration under
drought stress [1, 23]. Based on these conflicting reports,
it can be assumed that the interaction of Si and transpira-
tion mechanism might vary for different plant species/
genotypes, the nature of Si uptake and accumulation, and
13
Silicon
the environmental conditions. Different adaptive strategies
among species and genotypes within same species may act
to balance the amount of water absorbed and transpired. The
exact mechanisms behind these strategies, in responses to
Si, need to be further explored and addressed. Addition-
ally, even though lentil genotypes showed same trend in gas
exchange values (Pn, gs, Tr and Ci) for different treatments
in field and controlled conditions, lentil genotypes grown in
the field displayed comparatively higher values, compared
with the genotypes in the controlled conditions (Tables 3 and
4). This could be due to the variations in temperature, rela-
tive humidity, photoperiod, and soil properties under field
conditions.
4.3 Si Application Decreases the Canopy
Temperature (Tc) and Crop Water Stress Index
(CWSI) Values in Drought Stressed Lentil
Genotypes
Canopy temperature is considered to be a surrogate trait for
stomatal conductance, as they are directly related. Plants
with high stomatal conductance transpire more and thus
maintain a cooler IRTc. Drought stress increased IRTc for
all the genotypes and Si significantly decreased IRTc under
drought stress, giving further evidence of Si improving len-
til drought stress tolerance under both controlled and field
conditions. Lower IRTc, indicating better crop performance
through increased transpiration, has already been reported
in lentil genotypes [11]. These results showed that the
lower IRTc in Si treated plants could contribute to the Si
mediated plant water uptake, as revealed through increased
relative water content in leaves and enhanced water use effi-
ciency through efficient C ­ O2 fixation under drought stress
condition.
CWSI has been recognized as an indicator of plant water
status based on IRTc, Ta and RH. CWSI is essentially a ratio
of the difference between the actual canopy temperature and
non-stressed canopy temperature to the difference between
non-transpiring canopy temperature and non-stressed can-
opy temperature [54]. Thus, it is within expectations for the
control and Si treated plants to have a CWSI value of zero.
However, drought stress significantly increased CWSI in all
the genotypes. The maximum CWSI value is one, since IRTc
cannot be greater than the non—transpiring canopy tempera-
ture. Therefore, drought stressed lentil genotypes will have
CWSI values close to one [11]. The data is consistent with
this claim, with the drought susceptible genotype G3 hav-
ing the highest mean CWSI under drought stress, followed
by G2 and G1, under both controlled and field conditions.
Plants from the Si applied drought stress treatment signifi-
cantly moderated this increase and exhibited lower CWSI
values in comparison with the drought stressed genotypes
(Figs. 4 and 5).
Silicon
4.4 Si Enhances the Aboveground Biomass
and Yield Traits in Drought Stressed Lentil
Genotypes
Lentil genotypes from the field conditions exhibited low
biomass and yield, compared with the genotypes grown
under controlled conditions, although they shared the same
trend of change (increase/decrease) in different treatments.
This can be attributed to the difference in environmental
variables responsible for growth and yield of plants in the
field conditions. Shoot biomass has been associated with
both improved drought tolerance and increased root bio-
mass in lentils [55]. Decreased biomass production under
drought stress is consistent with the findings of Sehgal et al.
[5] in lentil genotypes. The aboveground biomass of all the
genotypes was significantly enhanced due to the applica-
tion of Si under drought stressed and non-stressed condi-
tions (controlled and field conditions), indicating improved
drought stress tolerance [16]. Si improved the performance
of drought susceptible and moderately tolerant genotypes to
moderately tolerant and drought tolerant, respectively. Thus,
it can be concluded that under drought stress, Si improved
the biomass production of all the genotypes irrespective of
their tolerance levels. Si also improved biomass production
of these genotypes in the non-stress environment but to a
lesser extent. There are conflicting reports on the effects of
Si under normal conditions, such as an increase in biomass
even under non-stressed conditions [56, 57] or no significant
differences in biomass [27, 58] However, the results of these
experiments suggest beneficial effects of Si to enhance the
aboveground biomass in lentil even without stress.
Increased growth hormone activity from Si application
might have also contributed to increased biomass production
under non-stressed conditions [56]. This Si mediated posi-
tive effect on biomass could also be attributed to its ability to
modify cell wall metabolism by improving tissue extensibil-
ity and enhancing cell enlargement [59]. Thus, the findings
clearly show the significant role of Si in inhibiting the dete-
rioration of cell membranes and improving rates of growth
and development of lentil under drought stress. The increase
in the values of yield related traits, in response to Si under
drought and non-stress conditions, clearly reflect improved
photosynthetic efficiency of lentil genotypes. Si supplemen-
tation to various crops, under drought stress and non-stress
conditions, have shown improved growth and grain yield in
potato [25], rice [60], and sugarcane [61].
4.5 Multivariate Data Analysis Shows The
Correlations Between Growth Room and Field
Experiments
Multivariate data analysis done in this study clearly dem-
onstrated the similarity patterns and correlations of the
studied parameters recorded from the controlled and field
experiments (Figs. 6a and 6b). The grouping patterns were
the same in both the biplots, which strongly validate and
express the same trend of results in both controlled and
field conditions. In both PCA biplots, as expected, drought
stressed genotypes moved away from the origin and were
positioned in the negative direction, showing less drought
stress tolerance based on the values of CWSI, IRTc and Tr.
However, with Si supplementation, under drought stress
(DSi), all the genotypes moved towards the positive direc-
tion, clearly showing an improvement in drought tolerance.
Furthermore, when Si was applied alone, without drought
stress (Si), all the genotypes moved closer to the genotypes
in drought stress supplemented with Si treatment group,
showing the positive effect of Si in improving the drought
tolerance levels of lentil genotypes, even under non-stress
condition. The genotypes in the control group were posi-
tioned near to the origin showing their normal behaviour.
Thus, this study clearly and strongly indicates the positive
role of Si in mitigating drought stress in lentil genotypes
and the improvement in photosynthetic efficiency, yield and
related traits under drought stress and non- stress conditions.
As obtained from the factor loadings (Table 2), PC1 was
mainly represented by Fv/Fm ratio, photosynthetic pigments,
Pn and gs in the positive side and IRTc and CWSI on the
negative side of the axis in the biplots obtained for both
controlled and field conditions. On the other hand, for the
biplot from the controlled conditions, PC2 was primarily
represented by Tr on the positive side and by IRTc on the
negative side of the axis, while in the biplot obtained from
the field, PC2 was characterised by IRTc on the positive side
and Tr on the negative side of the axis.
The two major clusters in the cluster diagram clearly
show the positive role of Si in mitigating drought stress by
clustering the Si supplemented drought stressed genotypes
along with genotypes from the control group (Fig. 7). Fur-
thermore, in the cluster diagram of controlled conditions,
the large cluster was clearly sub clustered in two small clus-
ters with genotypes from the drought stress supplemented
with Si treatment (DSi) in one and genotypes from control
(C) and Si alone (Si) treatments together in another clus-
ter. However, sub clustering was not observed in the cluster
diagram obtained from the field conditions. This might be
due to changes in various environmental conditions com-
pared to the controlled conditions in the growth chamber.
Correlation matrix revealed significant positive correlations
among and between chlorophyll pigments, Fv/Fm ratio, gas
exchange parameters (Pn, gs, Ci) and seed yield (Fig. 8). All
the above-mentioned traits showed significant negative cor-
relation with canopy temperature (IRTc) and CWSI. These
results also suggested a positive correlation of seed yield
with photosynthetic efficiency and negative correlation with
CWSI and transpiration rates (Tr). The results from the PCA,
13

cluster diagram and correlation matrix clearly showed that
Si supplementation has a positive role in alleviating drought
stress in lentil genotypes by enhancing photosynthetic effi-
ciency, yield and related traits.
5 Conclusion
Silicon supplementation under drought stress, in controlled
and field conditions, enhanced the photosynthetic potential
of lentil genotypes by protecting photosynthetic pigments
under drought stress. Chlorophyll fluorescence analysis sug-
gested that application of Si alleviated the adverse effects of
drought stress by increasing Fv/Fm ratio, thereby improv-
ing light use efficiency. Si also had ameliorative effects on
lentil growth and yield parameters under drought stress. In
addition, this study also validated the potential use of Si in
field conditions to mitigate drought stress in lentil plants,
without compromising the yield, based on improvements
in biomass production and seed yield as a direct result of
increased photosynthesis.
Acknowledgements The authors are grateful to the University of
Melbourne for the Australian Government Research Training Program
Scholarship and the Grains Research and Development Corporation
(GRDC), Australia for the Grain Industry Research Scholarship (GRS-
11011) given to Sajitha Biju.
Author Contributions Sajitha Biju: Study conception, design and
execution of experiments, sample preparation, methodology, data col-
lection, data analysis, prepared the first draft, reviewed, and edited the
draft, approved the final version.Sigfredo Fuentes: Study conception
and experiment design, reviewed the draft and approved the final ver-
sion.Dorin Gupta: Study conception and experiment design, reviewed
the draft and approved the final version.
Funding The research was supported by the University of Melbourne
(Australian Government Research Training Program Scholarship) and
the Grains Research and Development Corporation (GRDC), Australia
(Grain Industry Research Scholarship (GRS-11011).
Data Availability My manuscript and associated personal data.
Declarations
Ethics Approval Not applicable.
Consent to Participate Not applicable.
Consent for Publication Publishers can publish images and results,
there are no restrictions.
Competing Interests The authors declare no competing interests.
References
1. Chen W, Yao X, Cai K, Chen J (2011) Silicon alleviates
drought stress of rice plants by improving plant water status,
13
Silicon
photosynthesis and mineral nutrient absorption. Biol Trace Elem
Res 142:67–76. https://​doi.​org/​10.​1007/​s12011-​010-​8742-x
2. Ohashi Y, Nakayama N, Saneoka H, Fujita K (2006) Effects of
drought stress on photosynthetic gas exchange, chlorophyll fluo-
rescence and stem diameter of soybean plants. Biol Plant 50:138–
141. https://​doi.​org/​10.​1007/​s10535-​005-​0089-3
3. Falk S, Maxwell DP, Laudenbach DE, Huner NP (1996) Photo-
synthetic adjustment to temperature. In: Photosynthesis and the
environment. Springer, Dordrecht, pp 367–385. https://d​ oi.o​ rg/1​ 0.​
1007/0-​306-​48135-9_​15
4. Chaves MM (1991) Effects of water deficits on carbon assimila-
tion. J Exp Bot 42:1–6. https://​doi.​org/​10.​1093/​jxb/​42.1.1
5. Sehgal A, Sita K, Kumar J, Kumar S, Singh S, Siddique KH, Nay-
yar H (2017) Effects of drought, heat and their interaction on the
growth, yield and photosynthetic function of lentil (Lens culinaris
Medikus) genotypes varying in heat and drought sensitivity. Front
Plant Sci 8:1776. https://​doi.​org/​10.​3389/​fpls.​2017.​01776
6. Chaves MM, Pereira JS, Maroco J, Rodrigues ML, Ricardo CPP,
Osório ML, Carvalho I, Faria, T, Pinheiro C (2002) How plants
cope with water stress in the field? Photosynthesis and growth.
Ann Bot 89:907–916. https://​www.​jstor.​org/​stable/​42771​537
7. Li RH, Guo P, Michael B, Stefania G, Salvatore C (2006) Evalu-
ation of chlorophyll content and fluorescence parameters as indi-
cators of drought tolerance in barley. Agr Sci China 5:751–757.
https://​doi.​org/​10.​1016/​S1671-​2927(06)​60120-X
8. Ashfaq W, Brodie G, Fuentes S, Gupta D (2022) Infrared thermal
imaging and morpho-physiological indices used for wheat geno-
types screening under drought and heat stress. Plants 23:3269.
https://​doi.​org/​10.​3390/​plant​s1123​3269
9. Jones HG, Serraj R, Loveys BR, Xiong L, Wheaton A, Price AH
(2009) Thermal infrared imaging of crop canopies for the remote
diagnosis and quantification of plant responses to water stress in
the field. Funct Plant Biol 36:978–989. https://​doi.​org/​10.​1071/​
FP091​23
10. Fuentes S, De Bei R, Pech J, Tyerman S (2012) Computational
water stress indices obtained from thermal image analysis of
grapevine canopies. Irrig Sci 30:523–536. https://d​ oi.o​ rg/1​ 0.1​ 007/​
s00271-​012-​0375-8
11. Biju S, Fuentes S, Gupta D (2018) The use of infrared ther-
mal imaging as a non-destructive screening tool for identifying
drought-tolerant lentil genotypes. Plant Physiol Biochem 127:11–
24. https://​doi.​org/​10.​1016/j.​plaphy.​2018.​03.​005
12. Zargar SM, Mahajan R, Bhat JA, Nazir M, Deshmukh R (2019)
Role of silicon in plant stress tolerance: opportunities to achieve a
sustainable cropping system. 3 Biotech 9. https://d​ oi.o​ rg/1​ 0.1​ 007/​
s13205-​019-​1613-z
13. Ashfaq W, Fuentes S, Brodie G, Gupta D (2022b) The role of
silicon in regulating physiological and biochemical mechanisms
of contrasting bread wheat cultivars under terminal drought and
heat stress environments. Front Plant Sci 13. https://​doi.​org/​10.​
3389/​fpls.​2022.​955490
14. Liang Y, Sun W, Zhu YG, Christie P (2007) Mechanisms of
silicon-mediated alleviation of abiotic stresses in higher plants:
a review. Environ Pollut 147:422–428. https://​doi.​org/​10.​1016/j.​
envpol.​2006.​06.​008
15. Ma CC, Li QF, Gao YB, Xin TR (2004) Effects of silicon applica-
tion on drought resistance of cucumber plants. Soil Sci Plant Nutr
50:623–632. https://​doi.​org/​10.​1080/​00380​768.​2004.​10408​520
16. Epstein E (2009) Silicon: its manifold roles in plants. Ann Appl
Biol 155:155–160. https://​doi.​org/​10.​1111/j.​1744-​7348.​2009.​
00343.x
17. Biju S, Fuentes S, Gupta D (2017) Silicon improves seed germi-
nation and alleviates drought stress in lentil crops by regulating
osmolytes, hydrolytic enzymes and antioxidant defense system.
Plant Physiol Biochem 119:250–264. https://​doi.​org/​10.​1016/j.​
plaphy.​2017.​09.​001
Silicon
18. Biju S, Fuentes S, Gupta D (2021) Silicon modulates nitro-
oxidative homeostasis along with the antioxidant metabolism to
promote drought stress tolerance in lentil plants. Physiol Plant
172(2):1382–1398. https://​doi.​org/​10.​1111/​ppl.​13437
19. Biju S, Fuentes S, Gonzalez Viejo C, Torrico DD, Inayat S,
Gupta D (2021) Silicon supplementation improves the nutri-
tional and sensory characteristics of lentil seeds obtained from
drought-stressed plants. Sci Food Agric 101(4):1454–1466.
https://​doi.​org/​10.​1002/​jsfa.​10759
20. Zhu Y, Gong H (2014) Beneficial effects of silicon on salt and
drought tolerance in plants. Agron Sustain Dev 34:455–472.
https://​doi.​org/​10.​1007/​s13593-​013-​0194-1
21. Gong HJ, Chen KM, Chen GC, Wang SM, Zhang CL (2003)
Effects of silicon on growth of wheat under drought. J Plant
Nutr 26:1055–1063. https://​doi.​org/​10.​1081/​PLN-​12002​0075
22. Gong H, Zhu X, Chen K, Wang S, Zhang C (2005) Silicon alle-
viates oxidative damage of wheat plants in pots under drought.
Plant Sci 169:313–321. https://​doi.​org/​10.​1016/j.​plant​sci.​2005.​
02.​023
23. Gong H, Chen K (2012) The regulatory role of silicon on water
relations, photosynthetic gas exchange, and carboxylation activi-
ties of wheat leaves in field drought conditions. Acta Physiol Plant
1589–94 https://​doi.​org/​10.​1007/​s11738-​012-​0954-6
24. Pei ZF, Ming DF, Liu D, Wan GL, Geng XX, Gong HJ, Zhou
WJ (2010) Silicon improves the tolerance to water-deficit stress
induced by polyethylene glycol in wheat (Triticum aestivum L.)
seedlings. J Plant Growth Regul 29:106–115. https://​doi.​org/​10.​
1007/​s00344-​009-​9120-9
25. Crusciol CA, Pulz AL, Lemos LB, Soratto RP, Lima GP (2009)
Effects of silicon and drought stress on tuber yield and leaf bio-
chemical characteristics in potato. Crop Sci 49:949–954. https://​
doi.​org/​10.​2135/​crops​ci2008.​04.​0233
26. Gunes A, Pilbeam DJ, Inal A, Coban S (2008) Influence of silicon
on sunflower cultivars under drought stress, I: Growth, antioxidant
mechanisms, and lipid peroxidation. Commun Soil Sci Plant Anal
9:1885–1903. https://​doi.​org/​10.​1080/​00103​62080​21346​51
27. Shen X, Zhou Y, Duan L, Li Z, Eneji AE, Li J (2010) Silicon
effects on photosynthesis and antioxidant parameters of soybean
seedlings under drought and ultraviolet-B radiation. J Plant Phys-
iol 167:1248–1252. https://​doi.​org/​10.​1016/j.​jplph.​2010.​04.​011
28. Arnon (1949) Copper enzymes in isolated chloroplasts. Polyphe-
noloxidase in Beta vulgaris. Plant Physiol 24:1-15.https://​doi.​org/​
10.​1104/​pp.​24.1.1
29. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments
of photosynthetic biomembranes. Meth Enzymol 148:350–382.
https://​doi.​org/​10.​1016/​0076-​6879(87)​48036-1
30. Genty B, Briantais JM, Baker NR (1989) The relationship between
the quantum yield of photosynthetic electron transport and
quenching of chlorophyll fluorescence. Biochem Biophys Acta
Gen Subj 990:87–92. https://​doi.​org/​10.​1016/​S0304-​4165(89)​
80016-9
31. GRDC (Grains Research and Development Corporation) (2017)
Grow Notes Lentil Southern. www.​grdc.​com.​au.
32. Meena VD, Dotaniya ML, Coumar V, Rajendiran S, Kundu S,
Rao AS (2014) A case for silicon fertilization to improve crop
yields in tropical soils. Proc Natl Acad Sci India Sect B: Biol Sci
84:505–518. https://​doi.​org/​10.​1007/​s40011-​013-​0270-y
33. Matychenkov VV, Pinskiy DL, Bocharnikova A (1995) Influence
of mechanical compaction of soils on the state and form of avail-
able silicon. Eurasian Soil Sci 27:58–67
34. Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Dar-
nell J (2000) Photosynthetic stages and light-absorbing pigments.
Molecular Cell Biology. WH Freeman, NewYork
35. Baker NR, Oxborough K (2004) Chlorophyll fluorescence as
a probe of photosynthetic productivity. In: Papageorgiou GC,
Govindjee (eds) Chlorophyll a Fluorescence: A Signature of
Photosynthesis. Springer, Dordrecht, pp 65–82. https://​doi.​org/​
10.​1007/​978-1-​4020-​3218-9
36. Cruz de Carvalho MH (2008) Drought stress and reactive oxygen
species: production, scavenging and signaling. Plant Signal Behav
3:156–165. https://​doi.​org/​10.​4161/​psb.3.​3.​5536
37. Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant
machinery in abiotic stress tolerance in crop plants. Plant Physiol
Biochem 48:909–930. https://​doi.​org/​10.​1016/j.​plaphy.​2010.​08.​
016
38. Young AJ (1991) The photoprotective role of carotenoids in higher
plants. Physiol Plant 83:702–708
39. Kaufman PB, Takeoka Y, Carlson TE, Bigelow WC, Jones JD,
Moore PH, Ghosheh NS (1979) Studies on silica deposition in
sugarcane (Saccharum spp.) using scanning electron microscopy,
energy-dispersive X-ray analysis, neutron activation analysis, and
light microscopy. Phytomorphology 29:185–193
40. Wang Y, Zhang B, Jiang D, Chen G (2019) Silicon improves
photosynthetic performance by optimizing thylakoid membrane
protein components in rice under drought stress. Environ Exp Bot
158:117–124. https://​doi.​org/​10.​1016/j.​envex​pbot.​2018.​11.​022
41. Hattori T, Inanaga S, Araki H, An P, Morita S, Luxová M, Lux
A (2005) Application of silicon enhanced drought tolerance in
Sorghum bicolor. Physiol Plant 123:459–466. https://​doi.​org/​10.​
1111/j.​1399-​3054.​2005.​00481.x
42. Hattori T, Sonobe K, Inanaga S, An P, Morita S (2008) Effects
of silicon on photosynthesis of young cucumber seedlings under
osmotic stress. J Plant Nutr 31:1046–1058. https://​doi.​org/​10.​
1080/​01904​16080​19283​80
43. Habibi G, Hajiboland R (2013) Alleviation of drought stress by
silicon supplementation in pistachio (Pistacia vera L.) plants.
Folia Hortic 2:21–29. https://​doi.​org/​10.​2478/​fhort-​2013-​0003
44. Maghsoudi K, Emam Y, Ashraf M (2015) Influence of foliar
application of silicon on chlorophyll fluorescence, photosynthetic
pigments, and growth in water-stressed wheat cultivars differing
in drought tolerance. Turk J Bot 39:625–634. https://​doi.​org/​10.​
3906/​bot-​1407-​11
45. Cao BL, Ma Q, Xu K (2019) Silicon restrains drought-
induced ROS accumulation by promoting energy dissipation in
leaves of tomato. Protoplasma 7:1–1. https://​doi.​org/​10.​1007/​
s00709-​019-​01449-0
46 Farooq M, Wahid A, Kobayashi N, Fujita D, Basra SMA (2009)
Plant drought stress: effects, mechanisms and management. In:
Lichtfouse E, Navarrete M, Debaeke P, Véronique S, Alberola C
(eds) Sustainable Agriculture. Springer, Dordrecht, pp 153–188.
https://​doi.​org/​10.​1007/​978-​90-​481-​2666-8_​12
47. Jones HG (1985) Partitioning stomatal and non-stomatal limita-
tions to photosynthesis. Plant Cell Environ 8:95–104. https://​doi.​
org/​10.​1111/j.​1365-​3040.​1985.​tb012​27.x
48. Grassi G, Magnani F (2005) Stomatal, mesophyll conductance and
biochemical limitations to photosynthesis as affected by drought
and leaf ontogeny in ash and oak trees. Plant Cell Environ 28:834–
849. https://​doi.​org/​10.​1111/j.​1365-​3040.​2005.​01333.x
49. Agarie S, Agata W, Kubota F, Kaufman PB (1992) Physiological
roles of silicon in photosynthesis and dry matter production in
rice plants: I. Effects of silicon and shading treatments. Japanese
J Crop Sci 61:200–206
50. Jia W, Zhang J (2008) Stomatal movements and long-distance
signaling in plants. Plant Signal Behav 3:772–777. https://​doi.​
org/​10.​4161/​psb.3.​10.​6294
51. Chaves MM, Flexas J, Pinheiro C (2009) Photosynthesis under
drought and salt stress: regulation mechanisms from whole plant
to cell. Ann Bot 103:551–560. https://​d oi.​o rg/​1 0.​1 093/​a ob/​
mcn125
52. Gao X, Zou C, Wang L, Zhang F (2006) Silicon decreases transpi-
ration rate and conductance from stomata of maize plants. J Plant
Nutr 29:1637–1647. https://d​ oi.o​ rg/1​ 0.1​ 080/0​ 19041​ 60600​ 85149​ 4
13

53. Yoshida S (1965) Chemical aspects of the role of silicon in physi-
ology of the rice plant. Bull Natl Inst Agric Sci 15:18–58
54. Jackson RD, Idso SB, Reginato RJ, Pinter PJ Jr (1981)
Canopy temperature as a crop water stress indicator. Water
Resour Res 17:1133–1138. https://​d oi.​o rg/​1 0.​1 029/​W R017​
i004p​0 1133
55. Idrissi O, Houasli C, Udupa SM, De Keyser E, Van Damme
P, De Riek J (2015) Genetic variability for root and shoot
traits in a lentil (Lens culinaris Medik.) recombinant inbred
line population and their association with drought toler-
ance. Euphytica 204:693–709. https://​d oi.​o rg/​1 0.​1 007/​
s10681-​0 15-​1 373-8
56. Hamayun M, Sohn EY, Khan SA, Shinwari ZK, Khan AL,
Lee IJ (2010) Silicon alleviates the adverse effects of salin-
ity and drought stress on growth and endogenous plant
growth hormones of soybean (Glycine max L.). Pak J Bot
42:1713–1722
57. Shi Y, Zhang Y, Han W, Feng R, Hu Y, Guo J, Gong H (2016) Sili-
con enhances water stress tolerance by improving root hydraulic
conductance in Solanum lycopersicum L. Front Plant Sci 7:196.
https://​doi.​org/​10.​3389/​fpls.​2016.​00196
13
Silicon
58. Abdalla MM (2011) Beneficial effects of diatomite on growth,
the biochemical contents and polymorphic DNA in Lupinus albus
plants grown under water stress. ABJNA 2:207–220
59. Ma JF, Yamaji N (2006) Silicon uptake and accumulation in
higher plants. Trends Plant Sci 11:392–397. https://​doi.​org/​10.​
1016/j.​tplan​ts.​2006.​06.​007
60. Emam MM, Khattab HE, Deraz HNM, AE, (2014) Effect of
selenium and silicon on yield quality of rice plant grown under
drought stress. Aust J Crop Sci 8:596
61. Meyer JH, Keeping MG (2000) Review of research into the role
of silicon for sugarcane production. Proc South Afr Sugar Technol
Assoc 74:29–40
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