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ORIGINAL RESEARCH
published: 19 May 2017
doi: 10.3389/fpls.2017.00744
Frontiers in Plant Science | www.frontiersin.org 1May 2017 | Volume 8 | Article 744
Edited by:
P. V. Vara Prasad,
Kansas State University, USA
Reviewed by:
Djanaguiraman M.,
Kerala Students Union, India
Rohit Joshi,
Jawaharlal Nehru University, India
*Correspondence:
Harsh Nayyar
harshnayyar@hotmail.com
Specialty section:
This article was submitted to
Plant Abiotic Stress,
a section of the journal
Frontiers in Plant Science
Received: 07 January 2017
Accepted: 20 April 2017
Published: 19 May 2017
Citation:
Sita K, Sehgal A, Kumar J, Kumar S,
Singh S, Siddique KHM and Nayyar H
(2017) Identification of
High-Temperature Tolerant Lentil
(Lens culinaris Medik.) Genotypes
through Leaf and Pollen Traits.
Front. Plant Sci. 8:744.
doi: 10.3389/fpls.2017.00744
Identification of High-Temperature
Tolerant Lentil (Lens culinaris Medik.)
Genotypes through Leaf and Pollen
Traits
Kumari Sita 1, Akanksha Sehgal 1, Jitendra Kumar 2, Shiv Kumar 3, Sarvjeet Singh 4,
Kadambot H. M. Siddique 5and Harsh Nayyar 1*
1Department of Botany, Panjab University, Chandigarh, India, 2Indian Institute of Pulses Research, Kanpur, India,
3International Center for Agricultural Research in the Dry Areas, Rabat, Morocco, 4Department of Plant Breeding and
Genetics, Punjab Agricultural University, Ludhiana, India, 5The UWA Institute of Agriculture, The University of Western
Australia, Perth, WA, Australia
Rising temperatures are proving detrimental for various agricultural crops. Cool-season
legumes such as lentil (Lens culunaris Medik.) are sensitive to even small increases in
temperature during the reproductive stage, hence the need to explore the available
germplasm for heat tolerance as well as its underlying mechanisms. In the present
study, a set of 38 core lentil accessions were screened for heat stress tolerance by
sowing 2 months later (first week of January; max/min temperature >32/20◦C during
the reproductive stage) than the recommended date of sowing (first week of November;
max/min temperature <32/20◦C during the reproductive stage). Screening revealed
some promising heat-tolerant genotypes (IG2507, IG3263, IG3297, IG3312, IG3327,
IG3546, IG3330, IG3745, IG4258, and FLIP2009) which can be used in a breeding
program. Five heat-tolerant (HT) genotypes (IG2507, IG3263, IG3745, IG4258, and
FLIP2009) and five heat-sensitive (HS) genotypes (IG2821, IG2849, IG4242, IG3973,
IG3964) were selected from the screened germplasm and subjected to further analysis
by growing them the following year under similar conditions to probe the mechanisms
associated with heat tolerance. Comparative studies on reproductive function revealed
significantly higher pollen germination, pollen viability, stigmatic function, ovular viability,
pollen tube growth through the style, and pod set in HT genotypes under heat
stress. Nodulation was remarkably higher (1.8–22-fold) in HT genotypes. Moreover, HT
genotypes produced more sucrose in their leaves (65–73%) and anthers (35–78%) that
HS genotypes, which was associated with superior reproductive function and nodulation.
Exogenous supplementation of sucrose to in vitro-grown pollen grains, collected from
heat-stressed plants, enhanced their germination ability. Assessment of the leaves
of HT genotypes suggested significantly less damage to membranes (1.3–1.4-fold),
photosynthetic function (1.14–1.17-fold) and cellular oxidizing ability (1.1–1.5-fold) than
HS genotypes, which was linked to higher relative leaf water content (RLWC) and
stomatal conductance (gS). Consequently, HT genotypes had less oxidative damage
(measured as malondialdehyde and hydrogen peroxide concentration), coupled with a
higher expression of antioxidants, especially those of the ascorbate–glutathione pathway.
Controlled environment studies on contrasting genotypes further supported the impact
Sita et al. Heat Stress and Lentil Genotypes
of heat stress and differentiated the response of HT and HS genotypes to varying
temperatures. Our studies indicated that temperatures >35/25◦C were highly detrimental
for growth and yield in lentil. While HT genotypes tolerated temperatures up to 40/30◦C
by producing fewer pods, the HS genotypes failed to do so even at 38/28◦C. The
findings attributed heat tolerance to superior pollen function and higher expression of
leaf antioxidants.
Keywords: high temperature, reproductive growth, screening, tolerance, mechanisms, antioxidants
INTRODUCTION
Global temperatures would probably increase significantly by
the end of the twenty-first century due to anthropogenic
activities that will increase gases particularly carbon dioxide,
methane, chlorofluorocarbons, and nitrous oxides (Sánchez et al.,
2014). This will have devastating influence on the growth and
development of plants, further reducing their potential yield and
quality of food products (Delahunty et al., 2015). Moreover,
local increases in temperature are higher than the global level,
and more damaging to crops grown in these regions (Kaushal
et al., 2013; Kaur et al., 2015). The elevated temperatures,
particularly in tropical and subtropical regions, are markedly
affecting the growth and yield of various winter and summer-
season crops. These crops need to be examined as to how heat
stress affects their vegetative and reproductive growth stages
involving various morpho-physiological approaches. Various
studies on on legumes such as chickpea (Cicer arietinum L.;
Devasirvatham et al., 2012; Kaushal et al., 2013; Kumar et al.,
2013), pea (Pisum sativum L.;Guilioni et al., 1997), common
bean (Phaseolus vulgaris L.;Gross and Kigel, 1994), mung bean
(Vigna radiata L.; Tzudir, 2014) and cowpea (Vigna unguiculata
L.; Ahmed et al., 1992) have reported adverse effects of heat
stress. Similar studies on lentil are limited (Delahunty et al., 2015;
Bhandari et al., 2016; Kumar et al., 2016), where susceptibility
of vegetative growth and reproductive function to heat stress has
been depicted. Hence, further investigation is needed involving
a large number of contrasting genotypes grown under similar
heat stress environment to understand the mechanism of heat
tolerance in this crop.
Lentil is sown as a cool-season crop, and is highly susceptible
to rising temperatures. It needs low temperatures at the time of
vegetative growth, while maturity requires warm temperatures;
the best temperature for its optimum growth has been found
to be 18–30◦C (Sinsawat et al., 2004; Roy et al., 2012). Lentil is
also grown in relatively warmer regions in central and southern
parts of India, where the crop is exposed to supra-optimal
temperatures that reduce its yield potential (Verma et al., 2014).
Moreover, it has been observed that the chilling periods are
becoming shorter and the heat periods are becoming longer,
further resulting in exposure of cool-season crops to heat stress,
particularly in the reproductive stage (Hasanuzzaman et al.,
2013). Heat stress of 35◦C was vital in discriminating the
heat-tolerant and heat-sensitive genotypes in chickpea (Cicer
arietinum L.) and faba bean (Vicia faba L.) (Gaur et al., 2015).
Temperatures above 32/20◦C (max/min) during flowering and
pod filling in lentil can drastically reduce seed yield and quality
(Delahunty et al., 2015). In 2009, across southeastern Australia, a
heat wave (35◦C for 6 days) reduced the yield in lentil crops by
70% (Delahunty et al., 2015).
Heat stress can affect the growth, development, metabolism
and productivity of plants (Hasanuzzaman et al., 2013). Heat
stress causes various physiological changes in plants such
as leaf and stem scorching, leaf abscission and senescence,
shoot and root growth inhibition, reduction in the number of
flowers, inhibited pollen tube growth, pollen infertility, and fruit
damage, leading to catastrophic losses in crop yields (Bita and
Gerats, 2013; Teixeira et al., 2013; Hemantaranjan et al., 2014).
Above-normal temperatures also affect membrane stability,
water relations, photosynthesis, respiration and modulate
the concentration of hormones, and primary and secondary
metabolites (Hemantaranjan et al., 2014). In leaves, the process of
photosynthesis is recognized as susceptible to high temperatures
and may get retarded because of chlorosis, impaired electron
flow, thermolability of photosystem II (PSII), and decreased
carbon fixation as well as assimilation (Sinsawat et al., 2004).
Reproductive development (flowering and seed filling) is most
susceptible to high temperature stress; and rise in temperature
during flowering by a few degrees can lead to complete crop
loss (Wheeler et al., 2000; Asseng et al., 2011; Hatfield, 2011).
“At the time of reproduction, a brief phase of high temperature
may decrease the number of floral buds and augment abortion
of flowers abortion, significantly, though variations occur in
the response within and amid plant species as well as their
genotypes” (Annisa et al., 2013; Kaushal et al., 2013; Sage et al.,
2015). Investigations involving exposure to moderate heat stress
at various reproductive stages have associated development and
performance of pollen grains as being highly susceptible to
elevated temperature stress (Kaushal et al., 2013; Jiang et al., 2015;
Sage et al., 2015). High temperatures may interrupt reproductive
function by changing the concentrations of phytohormones like
auxins (Teale et al., 2006) and abscisic acid (Todaka et al., 2012).
Heat stress also speeds up the production and reactions
of reactive oxygen species (ROS) including singlet oxygen,
superoxide, hydroxyl radical and hydrogen peroxide thereby
inducing oxidative stress (Mittler, 2002; Hasanuzzaman
et al., 2012), which can significantly damage cell structure
(Chakraborty and Pradhan, 2012). Prolonged accumulation of
ROS is harmful and can inactivate enzymes, lipid peroxidation,
protein degradation and damage DNA (Chakraborty and
Pradhan, 2012). The plants have several enzymatic and non-
enzymatic systems, which detoxify the most toxic ROS to less
reactive molecules to limit oxidative damage under heat stress
(Sairam and Tyagi, 2004).
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Sita et al. Heat Stress and Lentil Genotypes
Enzymatic antioxidants, for example, peroxidase, catalase,
superoxide dismutase, ascorbate peroxidase as well as glutathione
reductase work for the removal of superoxides and hydrogen
peroxide (Mittler, 2002). Non-enzymatic antioxidants like
tocopherols, carotenoids, ascorbic acid, glutathione, also act
along with enzymatic antioxidants against oxidative stress (Foyer
and Noctor, 2003). Their higher expression has been linked with
heat tolerance in some previous studies lentil (Lens culinaris
Medik.; Chakraborty and Pradhan, 2011), soybean (Glycine max
L.; Devi and Giridhar, 2015), pea (Pisum sativum L.; Osman,
2015).
The mechanisms affecting heat tolerance are not fully known
in lentil. The objective of this study was to (a) screen the core
lentil germplasm for heat tolerance and (b) understand the basis
of heat tolerance using contrasting genotypes.
MATERIALS AND METHODS
Screening for Heat Tolerance
The seeds of 38 lentil (Lens culinaris Medik.) genotypes were
procured from different sources (Punjab Agricultural University,
Ludhiana, India; Indian Institute of Pulse Research, Kanpur,
India; ICARDA, Morocco). The lentil genotypes were sown in
earthen pots (8 kg capacity) on two sowing dates: (1) first week of
November 2013 for normal sowing and (2) first week of January
2014 for late sowing to impose heat stress at the reproductive
stage. For normal-sown plants, temperatures ranged from 27.3
to 6.1◦C (maximum) and 15 to 3◦C (minimum) during the
vegetative stage and from 31.5 to 17.4◦C (maximum) and 18.2 to
9.8◦C (minimum) during the reproductive stage. For late-sown
plants, temperatures ranged from 33 to 17.2◦C (maximum) and
18.2 to 9.2◦C (minimum) during the vegetative stage and from
38 to 27.5◦C (maximum) and 28–14◦C (minimum) during the
reproductive stage.
Relative humidity for normal-sown plants ranged from 100 to
72% (maximum) and 82 to 21% (minimum) during the vegetative
stage and from 95 to 77% (maximum) and 61 to 14% (minimum)
during the reproductive stage. In late-sown plants, the relative
humidity ranged from 97 to 61% (maximum) and 78 to 14%
(minimum) during the vegetative stage and from 93 to 42%
(maximum) and 59 to 12% (minimum) during the reproductive
stage.
Raising of Contrasting Genotypes
Based upon the response of the 38 lentil genotypes to heat
stress in the 2013–2014 trial above, several genotypes varying
in heat sensitivity (five heat-tolerant and five heat-sensitive)
were selected for further studies. These genotypes were sown
on two sowing dates: (1) 12 November 2014 for normal sowing
and (2) 15 January 2015 for late sowing to ensure heat stress
during reproductive growth. The plants were grown under
natural outdoor environment at Panjab University, Chandigarh,
India (30◦44′5.9994′′ N, 76◦47′27.5994′′ E). In northern
India, lentils are normally sown in November, the temperatures
throughout reproductive development remain below 32◦C/20◦C
(day time maximum/night time minimum); sowing in January
would ensure that plants were exposed to heat stress (above
32◦C/20◦C) at the time reproductive development. A sandy
loam soil (sand: 63.4%, silt: 24.6%, clay:12%) was mixed with
sand in a 3:1 ratio. The growth medium (soil) was prepared by
adding one part farmyard manure to three parts of the soil–
sand mixture. Ten mg kg−1of tricalcium phosphate fertilizer
was also added. The mixture was used to fill earthen pots (300
mm in diameter; 8 kg soil capacity) (Awasthi et al., 2014). For
inoculating the seeds, lentil-specific Rhizobium spp. was applied
prior to sowing. Initially, ten seeds were sown in each pot, upon
emergence, plants were thinned to five per pot, 15 days after
sowing (DAS).
Weather Data
The day time maximum/night time minimum and mean air
temperatures (Figure 1) and day time maximum/night time
minimum and mean relative humidity (Figure 1) were recorded
between 12 November 2014 and 5 May 2015. For normal-
sown plants, temperatures ranged from 31.8 to 10◦C (day time
maximum) and 17.8 to 6.2◦C (night time minimum) during the
vegetative stage and from 33.6 to 17◦C (day time maximum) and
21.6 to 8.2◦C (night time minimum) during the reproductive
stage. For late-sown plants, temperatures ranged from 33.6
to 17◦C (day time maximum) and 20.6 to 9.2◦C (night time
minimum) during the vegetative stage and from 39.2 to 25.6◦C
(day time maximum) and 25 to 15.7◦C (night time minimum)
during the reproductive stage (Figure 1).
Relative humidity (RH) for normal-sown plants ranged from
97 to 49% (day time maximum) and 90 to 19% (night time
minimum) during the vegetative stage and from 98 to 68% (day
time maximum) and 62 to 18% (night time minimum) during
the reproductive stage. In late-sown plants, the RH ranged from
98 to 68% (day time maximum) and 71 to 31% (night time
minimum) during the vegetative stage, and from 96 to 41% (day
time maximum) and 47 to 12% (night time minimum) during the
reproductive stage (Figure 1).
Photoperiod ranged between 11.2 and 11.4 h under normal-
sowing conditions, while under late-sown condtions, it ranged
from 12.2 to 12.4 h (Figure 1).
Thermal accumulation units were 1,218.5 in normal-sown
plants and 2,336.2 in late-sown plants. “Thermal units were
calculated as the total of the average temperatures of all previous
days until the initiation or completion of a particular stage”
(Awasthi et al., 2014).
Phenology, Biomass, and Yield
Components
The observations on phenology (days to flowering, podding and
maturity), biomass, flower number, pod set (%), pod number
and seed weight were recorded from 10 plants per genotype
in three replications (30 plants/genotype). “Observations from
replications for each genotype were pooled and averaged. The
flowers were tagged and examined for pod set. Yield-related traits
such as pod number, seed number and seed weight were also
recorded at maturity. For yield data, mature seeds were collected,
oven-dried for 3 days at 45◦C and then weighed, with average
values expressed on a per plant basis” (Awasthi et al., 2014).
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Sita et al. Heat Stress and Lentil Genotypes
FIGURE 1 | Temperature profile (◦C; top) and RH (%) and photoperiod (hours; bottom) during the normal sown and late sown experiments. Arrows indicate the
reproductive phase during both environments. Yellow line in the below figure represents the photoperiod during both sowing environments.
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Sita et al. Heat Stress and Lentil Genotypes
Reproductive Biology
For the analysis of reproductive function from contrasting
genotypes, flowers from individual plants (three per pot) were
collected randomly from five pots (15 plants/genotype) in three
replications from normal-sown and late-sown plants. The buds
or flowers were harvested from normal-sown plants when the
average temperature for the previous 7 days was lower than
25/19◦C and from late-sown plants when the corresponding
temperature was above 32/20◦C. Floral biology was examined as
follows:
Anther Morphology
The morphology of anthers was evaluated using scanning
electron microscopy (SEM). The fresh flowers were collected
early in the morning on the day of anthesis, from control and
stressed plants. Anthers of each genotype were collected from 10
flowers and put on a metallic stub. The anthers were observed
under SEM to examine any structural changes. “Anthers were
mounted fresh using double-stick tape, without dehydration,
and critical point drying, sputter coated with gold paladium
and scanned under SEM” (Postek et al., 1980; Kaushal et al.,
2013).
Pollen Morphology
“The morphology of pollen grains was studied using SEM. Pollen
grains were removed from the anthers (collected as described
above) and observed under SEM to examine any structural
changes. On the day of anthesis, fresh flowers were collected
early in the morning from control and stressed plants. Anthers
from 10 flowers were collected and teased on a metallic stub.
Samples were mounted fresh with double-stick tape, without
dehydration, and critical point drying, sputter coated with gold
paladium and scanned under SEM” (Postek et al., 1980; Kaushal
et al., 2013).
Pollen Viability
“About 200 pollen grains were tested for pollen viability was
tested on 200 pollen grains (5–10 microscopic fields) with 0.5%
acetocarmine/Alexander stain” (Kaushal et al., 2013). The pollen
grains were collected from flowers, which opened on the same
day. The pollen grains collected from flowers were pooled and
tested for their viability (Alexander, 1969). To select viable pollen
grains, section was made on the basis of size and shape (triangular
or spherical) of the pollen, and the concentration of the stain
taken up by the pollen (Kaushal et al., 2013).
Pollen Load and Pollen Germination
“The pollen load and pollen germination (in vivo) was tested
from the flowers (collected as above for pollen viability) with
fully-dehiscent anthers and pollen grains on the stigma. Pollen
load on the stigma was scored on a 1–5 scale (1 =low and 5
=high; Srinivasan et al., 1999). The number of germinated and
ungerminated pollen grains on the stigma surface were counted
from normal and heat-stressed flowers (Kaushal et al., 2013). The
in vitro pollen germination was tested in pollen grains collected
from five flowers per genotype in three replications. Pollen were
germinated as per the method of Brewbaker and Kwack (1963)
using a medium containing 10% sucrose, 1,640 mM boric acid,
1,269 mM calcium nitrate, 812 mM magnesium sulfate and 990
mM potassium nitrate (pH 6.5). Pollen grains were treated as
germinated when the size of tube exceeded the diameter of the
pollen grain. The percentage germination was determined from
at least 100 pollen grains per replicate” (Kaushal et al., 2013).
Pollen Germination (In vivo) and Fate of Pollen Tube
Growth
“The pollen germination on the stigma and pollen tube growth
through the style and in ovary was examined using Fluorescence
microscopy. The flowers were collected 1–3 days after anthesis
and fixed in acetic alcohol (1:3) for 24 h and then transferred to
8N NaOH for 6 h at 60◦C for clearing purposes. The complete
gynoecium part was transferred to aniline blue (0.1%), which was
kept overnight, followed by mounting on a slide in a 1:1 (aniline
blue: 10% glycerin) solution” (Kaushal et al., 2013). The stained
gynoecium was observed under a fluorescence photomicrograph
microscope (Nikon, Japan) (Dumas and Knox, 1983).
Stigma Receptivity
“To detect stigma receptivity, an esterase test was carried out
using a-naphthyl acetate as the substrate in the azo-coupling
reaction with fast blue B, as modified by Mattson et al. (1974).
Stigmas were removed from five flowers per genotype in three
replications 1 day before flower opening, immersed in a solution
containing a-naphthyl acetate and fast blue B in phosphate buffer,
at 37◦C for 15 min. The reddish brown color that developed on
the surface of the stigma was scored on a 1–5 scale (1 =low
receptivity and 5 =high receptivity)” (Kaushal et al., 2013).
Ovule Viability
Ovule viability was assessed via a 2, 3, 5-triphenyl-2 H-
tetrazolium chloride (TTC) reduction test. The ovules were
carefully removed from the ovary of five flowers per genotype in
three replications 1 day before anthesis. The ovules were placed
into a drop of TTC solution (0.5% TTC in 1% sucrose solution)
on a clean glass slide. The ovules were covered with a cover slip
and kept in a petri-dish having moist filer paper (2 layers). The
petri-dish was covered by a black paper and kept in the dark for
incubation at 25◦C in chamber. Following incubation of 15 min,
the ovules were examined under the microscope for viability,
which was measured on the basis of intensity of red color due
to formazan formation, especially in the central region. The red
color in the ovules is because of high availability of oxygen. The
intensity of the red color (ovule viability) was scored on a 1–5
scale (1 =lowest intensity and 5 =highest intensity) (Kaushal
et al., 2013).
To assess stress injury and biochemical traits, flowers and
subtending leaves were harvested randomly from five pots (15
plants/genotype) in three replications from normal-sown and
late-sown plants. The flowers or leaves were collected from
normal-sown plants when the average temperature for the
preceding 7 days was <25/19◦C, and from late-sown plants
(when the corresponding temperature was above 32/20◦C). The
following tests were conducted:
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Sita et al. Heat Stress and Lentil Genotypes
Stomatal Conductance and Leaf
Temperature
Stomatal conductance and leaf temperature of top fully-expanded
leaves were assessed with a portable leaf porometer (model SC1;
Decagon Devices, Pullman, WA, USA) (Kaushal et al., 2013).
Leaf Photosynthetic Function
“The photochemical efficiency of the intact leaves of the outdoor-
grown pants was measured as chlorophyll fluorescence using
the dark-adapted test of the modulated chlorophyll fluorometer
OS1-FL (Opti-Sciences, Tyngsboro, MA, USA). With this system,
chlorophyll fluorescence is excited by a 660 nm solid-state light
source with filters blocking radiation longer than 690 nm. The
average intensity of this modulated light was adjusted from 0 to 1
mE. Detection was in the 700–750 nm range using a PIN silicon
photodiode with appropriate filtering to remove extraneous light.
The clamps of the instrument were installed on the leaves to keep
them in the dark and to stop the light reaction of photosynthesis
for 45 min. After this, the clamps were attached to the optic fiber
of the device and the valves of the clamps were opened. After
starting the device, the 695 nm modulated light was radiated
through the optic fiber toward the leaf. Subsequently, the Fv/Fm
ratio was recorded. The leaves tested for chlorophyll fluorescence
were also used for measurement of chlorophyll concentration”
(Kaushal et al., 2013).
Membrane Damage (As Electrolyte
Leakage)
The leaves subtending the flowers were harvested from plants
grown under normal-sown and late-sown conditions (details as
above). Electrolyte leakage was used to assess the permeability
of the cell membrane as described by Lutts et al. (1996). Leaf
segments after washing with deionized water were placed in
closed vials containing deionized water (10 mL) and incubated
overnight at 25◦C. Electrical conductivity of the bathing solution
(C1) was determined after 24 h. Samples were then put in a
boiling water bath for 10–15 min and final conductivity reading
(C2) was obtained upon equilibration at 25◦C (Kaushal et al.,
2013). The electrolyte leakage (EL) was defined as follows:
EL% =C1/C2×100
Cellular Oxidizing Ability
The cellular oxidizing ability was measured as the 2, 3, 5-triphenyl
tetrazolium chloride (TTC) reduction ability (Steponkus and
Lanphear, 1967). One hundred mg of fresh leaf sample was
cut into small strips, immersed in incubation solution (50 mM
sodium phosphate, pH 7.4) containing TTC (500 mg 100 mL−1
solution) and incubated at 25◦C in the dark. Since the TTC
reduction is sensitive to excessive oxygen, the incubation of TTC
was carried out without shaking. After two extractions with 95%
ethanol (5 mL each), the extracts were pooled up to 10 mL.
Formazan, which forms in green tissues, was measured at 530
nm instead of 485 nm to avoid intervention by pigments such
as chlorophyll (Steponkus and Lanphear, 1967; Kaushal et al.,
2013). The observations were expressed as absorbance per g of
fresh weight (FW).
Chlorophyll Concentration
“To measure chlorophyll concentration, the fresh leaves (1.0 g)
were homogenized in 80% acetone, followed by centrifugation
at 5,701.8 g for 10 min. The absorbance of the supernatant was
recorded at 645 and 663 nm, and total chlorophyll was calculated
(Arnon, 1949) against 80% acetone as a blank” (Awasthi et al.,
2014). The chlorophyll content was measured as:
Chl a =12.9 (Abs663)−2.69 (Abs645)V
1, 000 ×W
Chl b =22.9 (Abs645)−4.68 (Abs663)V
1, 000 ×W
Total chl =Chl a +Chl b
where V is volume, W is tissue weight, Abs663 is absorbance at
663 nm and Abs645 is absorbance at 645 nm. The total chlorophyll
content was expressed as mg g−1DW.
Fresh tissue was collected for measuring chlorophyll
concentration and other biochemical traits but the calculations
were made on dry weight (DW) basis. The dry weight of the
fresh material was recorded by drying at 45◦C for 2 days. The
fresh weight was divided by the dry weight, the resultant vale
was multiplied with the calculated units of any biochemical
parameter.
Relative Leaf Water Content (RLWC)
RLWC was deterimined using the method of Barrs and
Weatherley (1962). Leaf tissue (100 mg) was collected from
control and stressed plants. The leaves were immersed in distilled
water for 2 h in a petri dish, removed, followed by surface drying
with blotters, and re-weighed (turgid weight, TW). The leaves
were then oven-dried at 110◦C for 24 h and weighed again for
dry weight (DW) The relative leaf water content was calculated
as follows:
Relative leaf water content (RLWC) =Fresh wt −Dry wt.
Turgid wt −Dry wt. ×10
Soluble Protein
The soluble protein concentration was estimated using a method
devised by Lowry et al. (1951). The plant material (100 mg) was
macerated in 0.1 M phosphate buffer (pH 7.0) and centrifuged
at 513.16 g for 15 min to obtain a supernatant. Five mL of TCA
(trichloroacetic acid; 15%) was added to the supernatant and kept
at 4◦C for 24 h. The mixture was then centrifuged at 513.16 g
for 15 min to separate the precipitates. The supernatant was
discarded and the precipitate dissolved in 0.1 N NaOH (1 mL),
kept for 18 h for complete dissolution, and treated as an extract.
To 1 mL of the above extract, copper sulfate reagent
containing 2% Na2CO3(in 0.1 N NaOH) and 0.5% CuSO4.5H2O
(in 1% sodium potassium tartrate) was added, allowed to stand
for 15 min, before adding 0.5 mL of Folin-phenol Ciocalteu’s
reagent (1:1 ratio) (1 N). This mixture was kept for 30 min for
color development before the absorbance was read at 570 nm.
The total protein content (mg g−1DW) was expressed using a
standard curve plotted with bovine serum albumin.
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Sita et al. Heat Stress and Lentil Genotypes
Sucrose
“The sucrose concentration was estimated as per the enzymatic
method given by Jones et al. (1977). Leaf tissue was homogenized
three times with 80% ethanol at 80◦C for 1.5 h for each extraction.
The extracts were pooled followed by evaporation at 40◦C in
an air-circulating oven. These were subsequently used to assay
sucrose concentration. Aliquots of 200 µL from standard sucrose
and the samples were added to 1 mL of reaction mixture
consisting of imidazole buffer 100 mM (pH 6.9; 40 mM imidazole
base, 60 mM imidazole-HCl), 0.4 mM NADP+, 1 mM ATP, 5
mM MgCl2, 0.5 mM dithiothreitol, 0.02% (w/v) bovine serum
albumin (BSA), 20 µg mL−1yeast invertase (E.C. 3.2.1.26), 2 µg
mL−1yeast hexokinase (E.C. 2.7.1.1) and 1 µg mL−1yeast P-
glucoisomerase (E.C. 5.3.1.9). The mixture was incubated at 25◦C
for 30 min to allow conversion of glucose and fructose to glucose
6-P. The absorption was meaured at 340 nm. Subsequently, 85
µL of glucose 6-P dehydrogenase (70 µl−1) was added, the
mixture was re-read after ∼5 min when absorbance became
constant. Blanks were run with 200 µL of extract and 1 mL of
the reaction mixture without invertase. The readings from each
sample were changed to sucrose concentration with a standard
curve” (Kaushal et al., 2013).
Sucrose Phosphate Synthase
To assay this enzyme, enzymes, the leaf tissue was homogenized
in a chilled HEPES buffer-NaOH (50 mM) pH 7 containing 2
mM MgCl2, 1 mM EDTA and 2 mM DTT as per the method
of Déjardin et al. (1997). The desalting of the supernatant was
done at 4◦C by passing it through 4 mL Sephadex G-25 columns
pre-equilibrated with buffer containing 20 mM HEPES–NaOH
(pH 7.5), 0.25 mM MgCl2, 0.01% 2-mercaptoethanol, 1 mM
ethylenediaminetetraacetic acid (EDTA) and 0.05% BSA. Sucrose
phosphate synthase (SPS) activity was assayed from this extract
following the anthrone test (Huber et al., 1989). During this
process, 70 mL of the reaction mixture including the extract was
adjusted to a final concentration of 4 mM Fru 6-P, 20 mM Glc 6-P,
3 mM UDPG, 50 mM HEPES–KOH (pH 7.5), 5 mM MgCl2and
1 mM EDTA. The reaction mixture was incubated at 37◦C for
15 min, and subsequently 70 mL of 30% (w/v) KOH was added
followed by heating for 10 min at 95◦C. To this, 1 mL of 0.14%
(w/v) anthrone (in 95% H2SO4) was added. The mixture was
incubated for 20 min at 37◦C, and absorbance recorded at 650
nm. The sucrose concentration was calculated from the standard
graph prepared with sucrose as the standard. The enzyme activity
was expressed as µg sucrose min−1mg−1protein.
Oxidative Molecules
Malondialdehyde
The damage to membranes was assessed on the basis of
a malondialdehyde (MDA) estimation, which indicates lipid
peroxidation of the membranes, using the method described by
Heath and Packer (1968). “Leaf tissue (1 g) was extracted in 10 mL
of 0.1% trichloroacetic acid (TCA), followed by centrifugation at
11,319.75 g for 15 min. Subsequently, 4 mL of 0.5% thiobarbituric
acid (in 20% trichloroacetic acid) was added to a 1-ml aliquot
of the supernatant. The final volume was 5 ml. Thereafter, the
mixture was heated for 30 min at 95◦C, and rapidly cooled in
an ice bath. After centrifugation at 5,701.8 g for 15 min, the
absorbance was measured at 532 nm. The value for non-specific
absorption at 600 nm was subtracted. MDA concentration was
calculated using its absorption coefficient of 155 mmol−1cm−1
and expressed as nmol g−1DW” (Kaushal et al., 2013).
Hydrogen Peroxide (H2O2)
The concentration of hydrogen peroxide (H2O2) was estimated
using the method of Mukherji and Chaudhari (1983). Plant tissue
(500 mg) was homogenized in 5 mL chilled acetone (80%) and
filtered through Whatman filter paper 1. Four mL of titanium
reagent was added followed by 5 mL of ammonia solution. The
mixture was centrifuged at 5,031 g for 15 min and the supernatant
was discarded. The residue was dissolved with 1 M H2SO4and
absorbance was recorded at 410 nm. The calculations were made
with a standard curve plotted with pure H2O2and expressed as
µmol g−1DW.
Antioxidants (Enzymatic and
Non-enzymatic)
Superoxide Dismutase
The activity of superoxide dismutase (SOD; E.C. 1.15.1.1) was
measured following the method of Dhindsa et al. (1981).
Fresh plant tissue was homogenized in 50 mM chilled/ice cold
phosphate buffer (pH 7.0) and centrifuged at 5,031 g for 15 min
at 4◦C and the supernatant was treated as an enzyme extract.
The reaction mixture (3 mL) contained 13 mM methionine,
25 mM nitro-blue-tetrazolium (NBT), 0.1 mM EDTA, 50 mM
sodium bicarbonate, 50 mM phosphate buffer (pH 7.8) and 0.1
mL of enzyme extract. The reaction was started by adding 2 mM
riboflavin and exposing to 15 W fluorescent light for 10 min.
The absorbance was recorded at 560 nm. SOD activity of the
samples was assayed by recording its capacity to decrease the
photochemical reduction of NBT. One unit of SOD activity was
defined as the amount of enzyme which causes 50% inhibition of
the photochemical reduction of NBT. It was expressed as Units
mg−1protein.
Catalase
Catalase (CAT; E.C. 1.11.1.6) activity was estimated using the
method of Teranishi et al. (1974) with some modifications.
The reaction mixture (3 mL) was prepared by mixing 50 mM
phosphate buffer (pH 7.0), 200 mM H2O2and 0.1 mL of enzyme
extract. The reaction was initiated by adding 200 mM H2O2.
The decrease in absorbance was recorded at 410 nm for 3 min.
Catalase activity was measured using the extinction coefficient 40
mM−1cm−1.
Ascorbate Peroxidase
Ascorbate peroxidase (APO; E.C. 1.11.1.11) activity was
determined by following the oxidation of ascorbate as a
reduction in absorbance at 290 nm, using the method of Nakano
and Asada (1981). Plant material was homogenized in ice cold
50 mM phosphate buffer, centrifuged at 5,031 g for 15 min at
4◦C and the supernatant was kept for assay. The reaction was
carried out at 20◦C in 3 mL of reaction mixture containing 50
mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbic
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Sita et al. Heat Stress and Lentil Genotypes
acid and enzyme extract. Ascorbic acid (2 mM) was added to
the reaction mixture to prevent inactivation of the enzyme. The
change in A290 was recorded at 30 s intervals after addition of
H2O2for 3 min. The rate constant was calculated using the
extinction coefficient of 2.8 mM−1cm−1.
Glutathione Reductase
Glutathione reductase (E.C. 1.6.4.2) was measured using the
method of Mavis and Stellwagen (1968). The reaction mixture
contained deionized water (0.65 mL), 100 mM phosphate buffer,
pH 7.6 (1.5 mL), glutathione oxidized (GSSG; 0.1 mL), β-
NADP (0.35 mL), BSA (0.20 mL) and enzyme solution (0.2
mL). The glutathione reductase enzyme solution was prepared
immediately before use and contained 0.30–0.60 units mL−1of
glutathione reagent in cold reagent, i.e., 1% BSA. The contents
of the reaction mixture were immediately mixed by inversion
and the reduction in absorbance was read at 340 nm for
approximately 3 min. The rate constant was calculated using the
extinction coefficient of 6.2 mM−1cm−1. The enzyme activity
was expressed as mmol oxidized donor min−1mg−1protein.
Ascorbic Acid
Ascorbic acid (AsA) was estimated according to the method of
Mukherji and Chaudhari (1983). Plant tissue was homogenized
in 6% TCA, and the homogenate was centrifuged at 3,649.15 g for
15 min. The supernatant was used as an extract for estimation.
Two mL of 2% DNPH (Dinitrophenylhydrazine) was added to
4 mL of extract, followed by one drop of 10% thiourea. The
mixture was boiled for 15 min in a water bath and then cooled to
room temperature; subsequently 5 mL of chilled sulfuric acid was
added at 0◦C. The absorbance was read at 530 nm, and ascorbic
acid concentration (mg g−1DW) was calculated from a standard
curve prepared by using known concentration ascorbic acid.
Glutathione
Reduced glutathione (GSH) was estimated following the method
of Griffith (1980). “Fresh leaf tissue was homogenized in 2 mL
of metaphosphoric acid followed by centrifugation at 14,539.59 g
for 15 min. The aliquots of the supernatant were neutralized by
adding 0.6 mL of 10% sodium citrate to 0.9 mL of the extract.
A total volume of 1 mL of assay containing 700 µl NADPH
(0.3 mM), 100 µl 5,5-dithio-bis-(2-nitrobenzoic acid (DTNB;
6 mM), 100 µl distilled water and 100 µl of the extract was
prepared and allowed to stabilize at 25◦C for 3–4 min. Thereafter,
10 µl of glutathione reductase (Sigma, USA) was added and
the absorbance recorded at 412 nm” (Kaushal et al., 2013).
Glutathione was calculated from a standard graph as described
by Griffith (1980) and expressed as nmol g−1DW.
Controlled Environment Studies
The contrasting genotypes of lentil (two heat-tolerant and
two heat-sensitive) were grown outdoors and, at the onset
of flowering (110–112 DAS) were subsequently subjected to
controlled environment conditions at varying heat stresss
[(35/25◦C, 38/28◦C, 40/30◦C; day time/night time); (RH: 65–75%
{day time}/45–55% {night time}] in a growth chamber (Meterx,
New Delhi, India). Pod number and seed weight per plant were
recorded. The plants were tested for reproductive function and
damage to leaves using various tests.
Effect of Sucrose on Pollen Germination (In vitro)
Pollen grains from heat-stressed flowers of two HT and two
HS genotypes were collected and germinated in a controlled
environment (method described above in reproductive biology)
in a growth medium supplemented with 1.0 and 2.5 µM sucrose.
The effects of sucrose concentration were tested on pollen
germination.
Statistical Analysis
There were 10 pots per genotype having 3 plants in 3 replications
for screening experiments, which were randomized following
RBD. For observations on contrasting genotypes too, 5 HT
and 5 HS genotypes were grown using RBD under normal and
late-won situations in 10 pots per genotype having 3 plants
in 3 replications. For CE studies, 3 pots having 3 plants were
used for each genotype using CRBD. Data were analyzed as
two factorial (temperature and genotypes) experimental design
using AGRISTAT statistical software (ICAR Research Complex,
Goa, India). Standard errors and least significant differences (P
<0.05) for genotypes, treatments and their interaction were
computed.
RESULTS
Field Experiment
In the initial experiment, 38 core accessions of lentil were
screened for heat tolerance at the reproductive stage on the basis
of biomass, filled pods per plant, seed weight per plant and
100-seed weight in 2013–2014.
Observations revealed that in normal-sown (NS) plants the
biomass ranged from 3.1 to 7.5 g plant−1across the screened
genotypes, but only 1.0–3.1 g plant−1in late-sown (LS) plants
due to heat stress (Table 1). Among all the genotypes, IG3327,
IG3312, IG3263, IG2507, IG3641, IG2458, IG4318, IG5146, and
FLIP2009 produced the most biomass (2.6–3.1 g plant−1) under
LS environment while IG2519, IG3568, IG4221, ILL6002, DPL15
produced the least (0.84–1.12 g plant−1).
Late-sown plants produced fewer filled pods (5.1–54.4
plant−1) due to heat stress than NS plants (25–127 plant−1)
(Table 1). Under LS conditions, the genotypes IG2507, IG3263,
IG3312, IG3327, IG3330, IG3546, IG3745, IG4258, IG5146,
and FLIP2009 produced the most filled pods (43–54.4 plant−1)
while IG2506, IG2510, IG2519, IG2802, IG2820, IG2878, IG3290,
IG3364, and IG4221 produced the least (5.1–9.4 plant−1).
Seed weight in NS plants ranged from 1.13 to 4.61 g plant−1
while LS plants ranged from 0.57 to 2.49 g plant−1(Table 1). In
LS plants, genotypes IG2507, IG3263, IG3297, IG3312, IG3327,
IG3330, IG3745, and FLIP2009 produced the heaviest seeds
(2.13–2.49 g plant−1) while IG2506, IG2510, IG2802, IG2820,
IG2878, IG3290, IG3364, IG3568, IG4221, ILL6002, DPL15, and
DPL315 produced the lightest seeds (0.57–0.91 g plant−1).
In NS plants, 100-seed weight (Table 1) ranged from 0.96
to 3.85 g plant−1but only 0.49–2.46 g plant−1in LS plants.
Genotypes IG2507, IG3263, IG3312, IG3546, IG4258, FLIP2009,
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Sita et al. Heat Stress and Lentil Genotypes
TABLE 1 | Biomass and yield components in normal-sown (NS) and late-sown (LS) lentil genotypes (Mean ±SE).
Genotypes Biomass g/plant Filled pods/plant Seed weight/plant 100 seed weight
NS LS NS LS NS LS NS LS
1 IG2506 4.3 ±0.8 0.87 ±0.18 38.9 ±3.9 5.1 ±1.3 1.51 ±0.24 0.57 ±0.16 1.22 ±0.13 0.65 ±0.11
2 IG2507 6.8 ±0.9 2.9 ±0.21 110 ±6.6 69.7 ±8.2 3.89 ±0.78 2.31 ±0.19 3.67 ±0.32 2.3 ±0.21
3 IG2510 5.4 ±0.8 1.8 ±0.13 49.4 ±5.3 9.4 ±1.9 1.81 ±0.67 0.76 ±0.16 1.61 ±0.29 1.1 ±0.17
4 IG2519 4.9 ±0.7 0.81 ±0.11 36.3 ±4.8 7.3 ±1.8 1.5 ±0.34 0.61 ±0.11 1.19 ±0.28 0.49 ±0.13
5 IG2802 5.8 ±0.9 1.7 ±0.13 41.5 ±4.4 7.5 ±1.9 1.83 ±0.23 0.81 ±0.13 1.65 ±0.17 0.97 ±0.15
6 IG2820 5.9 ±0.9 1.8 ±0.18 45.6 ±4.8 9.1 ±1.5 1.78 ±0.22 0.88 ±0.12 1.7 ±0.20 1.13 ±0.12
7 IG2821 6.1 ±0.8 2.1 ±0.15 83.5 ±5.5 18.9 ±2.2 3.88 ±0.26 1.09 ±0.14 1.97 ±0.23 0.7 ±0.11
8 IG2849 6.2 ±0.9 2.3 ±0.17 86.3 ±6.1 20.4 ±2.5 3.78 ±0.27 0.92 ±0.13 1.89 ±0.24 0.54 ±0.09
9 IG2878 5.7 ±0.8 1.7 ±0.13 41.4 ±4.5 9.3 ±1.3 1.76 ±0.24 0.77 ±0.14 1.66 ±0.21 0.94 ±0.17
10 IG3263 7.1 ±0.9 3.1 ±0.14 104 ±5.8 62.3 ±3.3 3.81 ±0.25 2.46 ±0.15 3.28 ±0.24 2.18 ±0.22
11 IG3290 5.8 ±0.8 1.6 ±0.16 44.3 ±4.9 9.3 ±1.4 1.71 ±0.22 0.78 ±0.15 1.63 ±0.26 0.89 ±0.21
12 IG3297 6.4 ±0.8 2.3 ±0.13 78.5 ±5.9 30.5 ±1.6 4.04 ±0.87 2.12 ±0.16 3.11 ±0.24 1.56 ±0.18
13 IG3312 7.3 ±0.9 2.8 ±0.24 111 ±8.9 43.8 ±1.5 4.32 ±0.82 2.11 ±0.19 3.46 ±0.28 2.01 ±0.23
14 IG3326 6.4 ±0.8 2.5 ±0.21 56.3 ±6.3 24.6 ±2.3 2.31 ±0.36 1.78 ±0.17 1.84 ±0.26 0.94 ±0.23
15 IG3327 7.2 ±0.8 2.7 ±0.19 121 ±6.8 48.3 ±2.3 4.14 ±0.38 2.19 ±0.18 3.44 ±0.25 1.91 ±0.29
16 IG3330 7.3 ±0.9 2.1 ±0.16 117 ±8.8 49.2 ±2.4 4.31 ±0.42 2.16 ±0.15 3.57 ±0.36 1.87 ±0.26
17 IG3364 5.3 ±0.9 1.4 ±0.14 53.2 ±6.6 9.4 ±1.8 1.79 ±0.24 0.81 ±0.14 1.68 ±0.27 1.9 ±0.29
18 IG3520 6.3 ±0.9 2.7 ±0.15 70.3 ±8.2 26.7 ±1.9 4.12 ±0.33 2.01 ±0.16 3.01 ±0.22 1.49 ±0.24
19 IG3537 6.1 ±0.8 2.8 ±0.16 73.4 ±8.4 28.7 ±1.5 4.08 ±0.37 2.09 ±0.17 3.09 ±0.28 1.67 ±0.28
20 IG3546 7.4 ±0.8 2.5 ±0.22 120 ±9.8 43.9 ±2.2 4.11 ±0.38 2.11 ±0.21 3.61 ±0.43 2.01 ±0.26
21 IG3568 4.8 ±0.8 0.84 ±0.13 35.6 ±6.8 6.1 ±1.3 1.59 ±0.28 0.65 ±0.14 1.17 ±0.26 0.56 ±0.19
22 IG3641 6.2 ±0.8 2.9 ±0.21 70.3 ±7.3 27.3 ±1.8 4.06 ±0.78 2.14 ±0.18 2.98 ±0.48 1.73 ±0.22
23 IG3745 7.5 ±0.9 2.6 ±0.23 127 ±9.5 74.5 ±5.4 3.79 ±0.82 2.49 ±0.16 3.79 ±0.62 2.21 ±0.24
24 IG3803 6.4 ±0.9 2.5 ±0.22 76.2 ±8.5 26.4 ±3.4 4.14 ±0.88 2.19 ±0.23 3.14 ±0.48 1.67 ±0.19
25 IG3964 5.14 ±0.8 1.45 ±0.11 78.4 ±9.4 28.7 ±3.5 2.98 ±0.63 0.89 ±0.18 2.34 ±0.43 0.819 ±0.22
26 IG3973 5.4 ±0.8 1.13 ±0.10 80.4 ±8.3 30.7 ±2.5 3.01 ±0.71 0.95 ±0.16 2.67 ±0.35 1.04 ±0.19
27 IG3984 6.6 ±0.8 2.6 ±0.24 66.4 ±9.2 28.9 ±3.5 4.11 ±0.91 2.11 ±0.24 3.06 ±0.56 1.71 ±0.21
28 IG4221 4.9 ±0.8 0.78 ±0.21 41.3 ±6.4 9.4 ±1.4 1.67 ±0.23 0.72 ±0.18 1.59 ±0.34 0.93 ±0.18
29 IG4242 5.4 ±0.8 1.78 ±0.18 81.7 ±5.9 24.6 ±2.5 3.67 ±0.31 1.11 ±0.17 1.79 ±0.44 0.71 ±0.19
30 IG4258 6.9 ±0.7 2.4 ±0.22 103 ±8.9 60.6 ±5.5 3.86 ±0.42 2.33 ±0.19 3.46 ±0.51 2.04 ±0.21
31 IG4318 6.2 ±0.9 2.68 ±0.25 80.3 ±7.4 34.5 ±4.3 4.02 ±0.46 2.13 ±0.21 3.13 ±0.48 1.77 ±0.22
32 IG5146 6.6 ±0.8 2.72 ±0.28 98 ±8.3 48.6 ±3.6 4.57 ±0.48 2.21 ±0.18 3.6 ±0.51 2.11 ±0.24
33 FLIP2009 6.8 ±0.8 2.4 ±0.22 103 ±6.3 48.9 ±3.2 3.83 ±0.51 2.3 ±0.18 3.85 ±0.46 2.48 ±0.25
34 DPL58 6.9 ±0.9 2.4 ±0.24 91.4 ±8.1 40.3 ±3.9 4.61 ±0.66 2.16 ±0.19 3.57 ±0.54 2.05 ±0.21
35 DPL315 5.2 ±0.6 1.67 ±0.19 40.3 ±4.5 19.4 ±3.5 2.17 ±0.35 0.95 ±0.16 1.81 ±0.51 0.85 ±0.18
37 DPL15 4.8 ±0.6 1.1 ±0.11 42.4 ±3.3 17.4 ±2.4 2.33 ±0.45 0.91 ±0.18 1.92 ±0.43 0.84 ±0.18
37 ILL-6002 3.04 ±0.7 0.985 ±0.14 25.3 ±2.3 13.4 ±1.5 1.13 ±0.24 0.82 ±0.17 0.96 ±0.21 0.77 ±0.19
38 DPL-15 4.7 ±0.7 1.12 ±0.12 48.6 ±2.7 31.6 ±2.6 2.23 ±0.27 1.11 ±0.20 2.01 ±0.38 0.89 ±0.17
LSD (P<0.05; genotype ×
date of sowing Interaction)
0.84 3.8 0.32 0.25
LSD, least significant difference.
and DPL58 had the highest 100-seed weights (2.04–2.46 g
plant−1) while IG2506, IG2519, IG2802, IG2878, IG3290,
IG3326, IG3568, IG4221, IG4242, DPL315, and DPL15 had the
lowest (0.64–0.97 g plant−1).
Based upon this screening, genotypes IG2507, IG3263,
IG3297, IG3312, IG3327, IG3546, IG3330, IG3745, IG4258,
and FLIP2009 were relatively tolerant to heat stress, whereas
genotypes IG2506, IG2519, IG2802, IG2821, IG2849, IG2878,
IG3290, IG3326, IG3568, IG3973, IG3964, IG4221, IG4242,
DPL315, and DPL15 were relatively heat-sensitive.
Of the 38 genotypes screened for heat tolerance, the five
most-tolerant (IG2507, IG3263, IG3745, IG4258, and FLIP2009)
and the five most-sensitive (IG2821, IG2849, IG4242, IG3973,
IG3964) to heat stress were selected for further studies to
probe the possible mechanisms related to heat tolerance. These
genotypes were subsequently grown in 2014–2015 and tested for
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Sita et al. Heat Stress and Lentil Genotypes
growth, yield, stress injury to leaves, and reproductive function
using various tests. The weather parameters (temperature, RH
and photoperiod) are detailed in the Materials and Methods
(Figure 1).
Growth and Yield
Flowering occurred in 108–112 DAS in NS plants and 51.2–53.4
DAS in LS plants (Table 2). Podding took 112–116 days to initiate
in NS plants and 54–59 days in LS plants (Table 2). The NS plants
matured in 140–145 days while the LS plants matured in 91–94
days (Table 2).
The flowering duration in LS plants was shorter than in NS
plants; by 10–11 days in heat-tolerant (HT) genotypes and 14–
18 days in heat-sensitive (HS) genotypes (Table 3). Similarly, the
podding duration was 9–11 days shorter in HT genotypes and
16–19 days in HS genotypes (Table 3).
Late-sown plants of HT genotypes produced 68–78 flowers per
plant compared to 106–140 in NS plants (Table 3). On the other
hand, late-sown plants of HS genotypes produced 35–49 flowers
per plant compared to 109–117 in NS plants. Pod set in LS plants
was 71–78% in HT genotypes and 32–41% in sensitive genotypes
(Table 3).
Late-sown HT genotypes produced 47–53% less biomass
than NS plants while HS genotypes showed produced 72–74%
less (Table 4). Similarly, the number of filled pods was 35–
40% less in late-sown HT genotypes and 60–66.8% less in
HS genotypes than NS plants. Seed weight per plant was 36–
41% less in HT genotypes and 55–70% less in HS genotypes.
One-hundred seed weight was 33–41% less in HT genotypes
and 61–70% less in HS genotypes. Genotype IG3745 produced
the highest biomass, pods/plant and seed weight/plant while
100-seed weight was highest in FLIP2009 under heat stress
environment (Table 4).
TABLE 2 | Phenology of various genotypes under normal-sown (NS) and late-sown (LS) plants (Mean ±SE); T, heat-tolerant, S, heat-sensitive.
Genotypes Days to flowering Days to podding Days to maturity
NS LS NS LS NS LS
IG2507 (T) 106 ±3.1a 53.2 ±2.4a 111 ±2.6a 55 ±1.5a 141 ±4.6a 91 ±6.9a
IG3263(T) 109 ±2.8a 51.3 ±2.3a 112 ±2.8a 57 ±1.9a 143 ±5.1a 90 ±8.9a
IG3745(T) 108 ±2.5a 52.4 ±2.1a 114 ±2.4a 58 ±1.5a 140 ±4.5a 94 ±9.2a
IG4258(T) 108 ±2.4a 53.2 ±2.3a 116 ±2.9a 54 ±1.6a 142 ±5.6a 92 ±8.4a
FLIP2009(T) 109 ±2.9a 51.2 ±2.4a 112 ±2.4a 56 ±1.8a 142 ±5.8a 91 ±9.1a
IG2821(S) 107 ±2.3a 53.3 ±2.2a 113 ±2.1a 57 ±1.4a 141 ±5.9a 93 ±6.5a
IG2849(S) 109 ±2.5a 52.3 ±2.5a 111 ±2.0a 59 ±1.3a 140 ±4.9a 94 ±5.8a
IG4242(S) 108 ±2.3a 53.4 ±2.3a 112 ±2.3a 56 ±1.5a 142 ±8.5a 92 ±5.9a
IG3973 (S) 107 ±2.2a 51.3 ±2.8a 114 ±2.5a 58 ±1.4a 140 ±9.3a 91 ±4.9a
IG3964 (S) 109 ±2.8a 52.2 ±2.1a 113 ±2.2a 56 ±1.6a 143 ±8.8a 93 ±5.1a
LSD (P<0.05; genotype ×
date of sowing Interaction)
3.4 2.9 3.3
Similar letters in a vertical column indicate no significant difference from each other. LSD, least significant difference.
TABLE 3 | Flowering and podding duration, number of flowers and pod set (%) in normal-sown (NS) and late-sown (LS) plants of heat-tolerant (T) and
heat-sensitive (S) genotypes (Mean ±SE).
Genotypes Flowering duration (days) Podding duration (days) Number of flowers Pod set (%)
NS LS NS LS NS LS NS LS
IG2507 (T) 28 ±2.3a 18 ±2.7a 30 ±2.6a 20 ±1.5a 123 ±4.6ab 78 ±6.9a 89.4 ±6.8a 78.4 ±8.4a
IG3263(T) 30 ±3.1a 20 ±2.1a 28 ±2.8a 19 ±1.9a 118 ±5.1ab 71 ±8.9a 88.4 ±8.7a 77.7 ±8.3a
IG3745(T) 31 ±2.3a 20 ±1.9a 30 ±2.4a 20 ±1.5a 140 ±4.5a 82 ±9.2a 90.7 ±9.3a 71.2 ±8.1a
IG4258(T) 29 ±2.5a 18 ±2.5a 30 ±2.9a 19 ±1.6a 106 ±5.6c68 ±8.4ab 84 ±7.7a 76.1 ±8.3a
FLIP2009(T) 30 ±3.3a 20 ±2.2a 29 ±2.4a 18 ±1.8a 131 ±5.8a 69 ±9.1ab 88.9 ±8.4a 77.8 ±8.2a
IG2821(S) 29 ±2.5a 14 ±1.1b 28 ±2.1a 12 ±1.4b 117 ±5.9ab 37 ±6.5c71.3 ±9.4b 41 ±5.6b
IG2849(S) 31 ±2.8a 13 ±1.6b 29 ±2.0a 11 ±1.3b 124 ±4.9ab 35 ±5.8c69.5 ±8.6b 40 ±4.5b
IG4242(S) 28 ±2.6a 12 ±1.7b 29 ±2.3a 10 ±1.5b 115 ±8.5ab 40 ±5.9c71 ±9.5b 38 ±4.1b
IG3973 (S) 29 ±2.3a 11 ±1.3b 30 ±2.5a 12 ±1.4b 113 ±9.3ab 48 ±4.9c71 ±9.2b 32 ±4.8b
IG3964 (S) 30 ±3.2a 12 ±1.2b 26 ±2.2a 10 ±1.6b 109 ±8.8c49 ±5.1c71.5 ±8.9b 37 ±4.3b
LSD (P<0.05; genotype ×
date of sowing Interaction)
3.4 2.9 10.3 10.5
Similar letters in a vertical column indicate no significant difference from each other. LSD, least significant difference.
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Sita et al. Heat Stress and Lentil Genotypes
TABLE 4 | Yield components in normal-sown (NS) and late-sown (LS) plants of heat-tolerant (T) and heat-sensitive (S) genotypes (Mean ±SE).
Genotypes Biomass/plant Filled pods/plant weight Seed weight/plant 100 seed weight
NS LS NS LS NS LS NS LS
IG2507 (T) 6.4 ±0.43a 3.07 ±0.35a 110 ±4.8b 69.7 ±8.5a 3.89 ±0.68a 2.31 ±0.36a 3.67 ±0.32a 2.3 ±0.14a
IG3263(T) 6.9 ±0.36a 3.2 ±0.41a 104 ±5.9b 62.3 ±9.2b 3.81 ±0.71a 2.46 ±0.33a 3.28 ±0.36c 2.18 ±0.18b
IG3745(T) 7.2 ±0.41a 3.8 ±0.37a 127 ±8.3a 74.5 ±7.8a 3.79 ±0.73a 2.49 ±0.36a 3.79 ±0.42a 2.21 ±0.15b
IG4258(T) 7.3 ±0.45a 3.2 ±0.38a 90 ±9.1c 58.6 ±8.3b 3.86 ±0.74a 2.33 ±0.37a 3.46 ±0.35b 2.04 ±0.15b
FLIP2009(T) 7.1 ±0.44a 3.4 ±0.42a 103 ±9.4b 60.6 ±7.7b 3.83 ±0.81a 2.3 ±0.31a 3.85 ±0.37a 2.46 ±0.13a
IG2821(S) 6.7 ±0.48a 1.9 ±0.23b 83.5 ±8.7c 18.9 ±3.2c 3.21 ±0.78a 1.09 ±0.35b 1.97 ±0.28f 0.7 ±0.14cd
IG2849(S) 6.5 ±0.54a 1.8 ±0.35b 86.3 ±9.1c 20.4 ±2.7c 3.11 ±0.81a 0.92 ±0.23b 1.89 ±0.21f 0.54 ±0.10cd
IG4242(S) 6.1 ±0.47a 1.6 ±0.25b 81.7 ±8.9c 24.6 ±4.3c 3.26 ±0.68a 1.11 ±0.21b 1.79 ±0.24f 0.71 ±0.11cd
IG3973 (S) 5.9 ±0.51ab 1.3 ±0.28b 80.4 ±9.3c 30.7 ±3.1c 3.01 ±0.63a 0.95 ±0.27b 2.67 ±0.23d 1.04 ±0.12c
IG3964 (S) 6.2 ±0.42a 1.8 ±0.29b 78.4 ±8.4cd 28.7 ±2.8c 3.19 ±0.71a 0.89 ±0.18b 2.34 ±0.25e0.82 ±0.12d
LSD (P<0.05; genotype ×
date of sowing Interaction)
1.2 10.5 0.46 0.21
Similar letters in a vertical column indicate no significant difference from each other. LSD, least significant difference.
The number of nodules ranged from 10.8 to 13.1 in NS plants
(Table 5). Late-sown plants produced 38–44% fewer nodules in
HT genotypes and 68–75% fewer in HS genotypes. HT genotypes
IG4258 and IG3745 produced the most nodules (7.4) whereas HS
genotypes IG3973 and IG2849 produced the least (2.9).
Status of Damage to Leaves Due to Heat
Stress
Leaf temperatures ranged from 29 to 31◦C in NS plants, while
in LS plants, leaf temperatures varied from 32.9 to 34.7◦C in HT
genotypes and 37.5–38.7◦C in HS genotypes Genotype FLIP2009
showed the lowest leaf temperature (32.9◦C) under heat stress
(Table 6).
The stomatal conductance (gS) of leaves ranged from 319 to
424 mmol m−2s−1in NS plants. It was 205–313 mmol m−2s−1
in LS plants of HS genotypes and 390–497 mmol m−2s−1in HT
genotypes (Table 6). Genotype FLIP2009 had the highest gS in
the LS environment (Table 6).
Leaf water status was measured using relative leaf water
content (RLWC), which varied from 78.3 to 86.4% in NS
plants (Table 6). Late-sown plants had lower RLWCs, but it
was relatively higher in HT genotypes (73.4–82.6%) than HS
genotypes (62–65%). A HT genotype IG4258 had the highest
RLWC (74.6%) when late-sown, while an HS genotype (IG4242)
had the lowest (62.8%).
Membrane damage was recorded as electrolyte leakage (EL),
which ranged from 12.5 to 15.3% (Table 7). In LS plants, EL
increased due to heat stress; HT genotypes had less damage (18.4–
20.3%) than HS genotypes (21.3–25.5%). Genotypes IG3263 and
FLIP2009 had the least damage while IG2821 and IG4242 had the
most.
Photosynthetic efficiency was measured as PSII function
(Fv/Fm ratio). In LS plants, HS genotypes showed more
reduction (21–26%) in PSII function than HT genotypes (10–
15% reduction) (Table 7). A HT genotype IG3263 had the highest
PSII function (0.71) while the HS genotype IG3964 had the
lowest (0.58).
TABLE 5 | Number of nodules in in normal-sown (NS) and late-sown (LS)
plants of heat-tolerant (HT) and heat-sensitive (HS) genotypes (Mean ±
SE).
Genotypes Nodule number
NS LS
IG2507 (T) 11.3 ±2.1a 6.4 ±0..67a
IG3263(T) 12.5 ±1.8a 6.9 ±0.71a
IG3745(T) 13.1 ±1.6a 7.2 ±0.64a
IG4258(T) 12.1 ±1.9a 7.4 ±0.58a
FLIP2009(T) 11.8 ±1.5a 6.8 ±0.66a
IG2821(S) 10.8 ±1.8a 3.4 ±0.62b
IG2849(S) 12.4 ±1.7a 2.9 ±0.69b
IG4242(S) 12.6 ±1.9a 3.6 ±0.61b
IG3973 (S) 11.9 ±1.7a 2.9 ±0.71b
IG3964 (S) 12.3 ±1.8a 3.1 ±0.62b
LSD (P<0.05; genotype ×
date of sowing Interaction)
2.1
Similar letters in a vertical column indicate no significant difference from each other. LSD,
least significant difference.
Stay-green trait was measured as the loss of total chlorophyll
(chl) in leaves (Table 7). Total chlorophyll concentration in LS
plants was lower (4.8–13.1 mg g−1DW) than NS plants (13.8–
15.6 mg g−1DW). In LS plants, HT genotypes retained more
total chlorophyll (10.8–13.1 mg g−1DW) than HS genotypes
(4.8–9.3 mg g−1DW). Under heat stress, HT genotype IG3263
had the most chl (13.1 mg g−1DW) while HS genotype IG4242
had the least (4.8 mg g−1DW) chlorophyll.
Cellular oxidizing ability was measured in a TTC reduction
assay (Table 7). Late-sown plants had significantly less cellular
oxidizing ability (14–54%) than NS plants. HT genotypes
maintained higher oxidizing ability values (0.17–0.2 units) than
HS genotypes (0.11–0.13 units) under heat stress. Genotype
IG2507 maintained highest respiration ability under hear stress.
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Sita et al. Heat Stress and Lentil Genotypes
TABLE 6 | Leaf temperature, stomatal conductance (gS) and relative leaf water content (RLWC) in normal-sown (NS) and late-sown (LS) plants of
heat-tolerant (HT) and heat-sensitive (HS) genotypes (Mean ±SE).
Genotypes Leaf temperature (◦C) Stomatal conductance (gS) mmol/m−2/s−1RLWC (%)
NS LS NS LS NS LS
IG2507 (T) 30.6 ±0.35a 33.4 ±0.23b 319.4 ±9.7b 390.5 ±10.9d 79.4 ±3.2b 73.4 ±3.3c
IG3263(T) 31.4 ±0.23a 34.7 ±0.18b 323.5 ±11.3b 410.4 ±12.5c 83.5 ±4.2a 80.4 ±4.1a
IG3745(T) 29.8 ±0.21a 33.5 ±0.17b 420.6 ±12.6a 489.3 ±12.5a 86.4 ±2.9a 82.6 ±3.8a
IG4258(T) 30.4 ±0.24a 34.7 ±0.20b 327.2 ±13.5b 443.5 ±11.9b 79.5 ±3.1b 74.8 ±3.5c
FLIP2009(T) 29.6 ±0.22a 32.9 ±0.19bc 421.1 ±11.9a 497.5 ±13.9a 82.3 ±2.8a 78.4 ±3.7b
IG2821(S) 30.9 ±0.15a 38.4 ±0.23a 317.4 ±12.8b 221.3 ±12.7f 82.5 ±4.2a 63.2 ±2.8d
IG2849(S) 31.5 ±0.18a 37.4 ±0.25a 319.4 ±13.6b 205.6 ±11.9f 80.4 ±3.8b 65.1 ±3.1d
IG4242(S) 29.6 ±0.19a 37.6 ±0.26a 424.5 ±14.3a 313.5 ±14.5e 83.4 ±4.5a 62.8 ±4.2d
IG3973 (S) 30.8 ±0.20a 38.7 ±0.21a 326.5 ±12.3b 210.5 ±13.8g 78.9 ±3.7b 63.2 ±3.6d
IG3964 (S) 29.7 ±0.22a 37.5 ±0.25a 320.5 ±13.1b 224.5 ±16.7f 81.3 ±4.2b 64.2 ±4.1d
LSD (P<0.05; genotype ×
date of sowing Interaction)
1.3 19.5 3.9
Similar letters in a vertical column indicate no significant difference from each other. LSD, least significant difference.
TABLE 7 | Electrolyte leakage, Photosystem (PS) II function, Chlorophyll (Chl) concentration and 2,3,5-Triphenyl tetrazolium chloride (TTC) reduction
ability in normal-sown (NS) and late-sown (LS) plants of heat-tolerant (HT) and heat-sensitive (HS) genotypes (Mean ±SE).
Genotypes Electrolyte leakage (%) PS II (Fv/Fm ratio) Chlorophyll (mg/g DW) TTC reduction ability
(Absorbace530/g FW)
NS LS NS LS NS LS NS LS
IG2507 (T) 13.5 ±2.5a 19.4 ±2.6b 0.8 ±0.04a 0.68 ±0.06a 15.6 ±1.9a 12.4 ±1.9a 0.23 ±0.03a 0.19 ±0.03a
IG3263(T) 12.8 ±2.9a 18.4 ±2.1b 0.79 ±0.03a 0.71 ±0.07a 16.8 ±2.1a 13.1 ±1.7a 0.21 ±0.04a 0.17 ±0.03a
IG3745(T) 14.3 ±2.6a 19.5 ±1.8b 0.8 ±0.05a 0.69 ±0.05a 14.7 ±1.8a 11 ±1.7a 0.25 ±0.05a 0.18 ±0.02a
IG4258(T) 15.3 ±2.8a 20.3 ±1.6b 0.78 ±0.04a 0.7 ±0.06a 14.9 ±1.7a 10.8 ±1.6a 0.24 ±0.03a 0.2 ±0.02a
FLIP2009(T) 12.4 ±1.8a 18.4 ±1.5b 0.8 ±0.05a 0.68 ±0.08a 15.1 ±1.8a 11.7 ±1.8a 0.21 ±0.03a 0.18 ±0.02a
IG2821(S) 13.2 ±2.5a 24.3 ±2.2a 0.8 ±0.06a 0.59 ±0.08b 14.9 ±1.9a 7.9 ±1.3b 0.23 ±0.04a 0.11 ±0.03b
IG2849(S) 12.7 ±2.4a 23.4 ±2.8a 0.8 ±0.05a 0.56 ±0.07b 15.6 ±1.7a 8.3 ±1.5b 0.22 ±0.05a 0.13 ±0.02b
IG4242(S) 14.1 ±2.8a 25.5 ±2.9a 0.78 ±0.05a 0.58 ±0.08b 14.5 ±1.7a 4.8 ±1.2d 0.24 ±0.03a 0.13 ±0.02b
IG3973 (S) 12.5 ±2.9a 21.3 ±2.7a 0.79 ±0.07a 0.52 ±0.06b 16.3 ±1.8a 9.3 ±1.2b 0.21 ±0.05a 0.12 ±0.03b
IG3964 (S) 13.2 ±2.5a 23.4 ±2.6a 0.8 ±0.08a 0.58 ±0.08b 13.8 ±1.6ab 6.9 ±1.3c0.24 ±0.03a 0.11 ±0.02b
LSD (P<0.05; genotype ×
date of sowing Interaction)
3.1 0.084 1.92 0.04
Similar letters in a vertical column indicate no significant difference from each other. LSD, least significant difference; Fv, variable fluorescence; Fm, maximum fluorescence.
TEM studies of leaves from heat-stressed plants showed cell
wall thickening, severe damage to chloroplasts (as indicated
by their shrinkage), fewer and smaller starch grains in the
chloroplast, the disintegration of the chloroplast envelope, and
damage to granal thylakoids, more so in HS genotypes (Figure 2,
arrows). Late-sown HT genotypes had fewer mitochondria than
late-sown HS genotypes Figure 2n arrows. The mitochondria
also swelled under heat stress from (recorded as increase in
size from 0.5 to 1.60 µm) as shown in Figures 2m,n arrows,
while the nucleus contracted and the nucleus showed dispersed
chromatin, with more damage to HS genotypes (Figure 2,
arrows).
Oxidative damage was measured as malondialdehyde (MDA)
and hydrogen peroxide concentration (Figure 3) in the leaves
of NS and LS plants. MDA concentration is an indicator of
oxidative damage to membranes, which was 1.6–2.7-fold higher
in LS plants than in NS plants; HT genotypes showed significantly
less damage (1.64–1.8-fold) than HS genotypes (2.1–2.7-fold).
Hydrogen peroxide followed a similar trend in LS plants and
showed 1.4–1.7-fold increase in HT genotypes and 2.1–2.8-
fold increase in HS genotypes. Genotypes IG2507 and FLIP
2009 exhibited lowest damage as MDA and hydrogen peroxide
concentrations, respectively.
Various antioxidants (enzymatic and non-enzymatic) were
tested in leaves. Late-sown plants produced 25–47% more
superoxide dismutase (SOD) than NS plants (Figure 4). Two
HS genotypes (IG2849 and IG3964) produced significantly
more SOD (47–48%) than the other genotypes. The differences
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Sita et al. Heat Stress and Lentil Genotypes
FIGURE 2 | Comparative TEM observations of the ultra-structure of leaves in normal sown (NS) and late sown (LS) plants. Cell organelles from (a,b) NS
and (c,d) LS plants; chloroplast from (e,f) NS and (g,h) LS plants; nucleus of (i,j) NS and (k,l) LS plants; mitochondria (m,n) from LS plants; cell wall (o,p) from LS
plants. Arrows indicate (b,d) disrupted cell organelles, (p) thickened cell wall, (f,h) disrupted chloroplast, (m,n) damaged and increased number of mitochondria, (p)
dispersed chromatin in nucleus, (d,p) shrinkage of vacuoles, and (d,h) fewer and smaller starch granules in the chloroplast in a heat-sensitive genotype under LS
conditions.
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Sita et al. Heat Stress and Lentil Genotypes
FIGURE 3 | (A) Malondialdehyde (MDA) and (B) hydrogen peroxide (H2O2) concentration in normal-sown (NS) and late-sown (LS) lentil genotypes. Small vertical bars
represent standard errors. LSD (P<0.05; represented as small vertical line close to y-axis) for MDA (2.9) and H2O2(0.83); T, tolerant; S, sensitive.
between HT and HS genotypes for SOD activity were
small.
Catalase increased more (31–95%) than SOD (25–47%) in LS
plants (Figure 4). The genotypes IG2507 (HT) and IG3964 (HS)
had the most enzyme activity while genotype IG2821 (HS) had
the least.
Ascorbate peroxidase activity was 27–40% higher in LS
plants of HT genotypes while it was 25–32% lesser in HS
genotypes (Figure 4). Genotypes IG4258 (HT) and FLIP2009
(HT) produced the most APX under LS conditions.
Late-sown HT genotypes produced 23–44% more glutathione
reductase than NS plants, while HS genotypes produced 22–43%
less (Figure 4). GR activity showed highest increase in a HT
genotype IG2507 under heat stress.
Late-sown HT genotypes produced 15–22% more ascorbate
(ASC) than NS plants, while it was found to be 13–21% less in
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Sita et al. Heat Stress and Lentil Genotypes
FIGURE 4 | (A) Superoxide dismutase (SOD), (B) catalase (CAT), (C) ascorbate peroxidase (APX), and (D) glutathione reductase (GR) in normal-sown (NS) and
late-sown (LS) genotypes. Small vertical bars on histograms represent standard errors. LSD (P<0.05; represented as small vertical line close to y-axis) for SOD (1.2),
CAT (1.6), APX (1.4), and GR (1.8); T, tolerant; S, sensitive.
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Sita et al. Heat Stress and Lentil Genotypes
HS genotypes (Figure 5). A HT genotype IG3745 had the highest
increase in ASC concentration (22%) under LS conditions.
Reduced glutathione (GSH) concentration increased more
than ASC concentration under heat stress. Late-sown HT
genotypes produced 38–53% more GSH than NS plants, while HS
genotypes produced 27–35% less (Figure 5). Highest GSH was
found in Genotype IG3745 under heat stress environment.
Sucrose and Sucrose Phosphate Synthase
Activity in Leaves and Anthers
Late-sown plants significantly had less sucrose concentration in
both the anthers and leaves than NS plants (Figure 6). Normal-
sown plants had 24.3–31.3 mg g−1DW sucrose in leaves and
17.9–21.5 mg g−1DW in anthers compared with LS plants which
had 10.6–21.6 mg g−1DW in leaves and 7.5–13.4 mg g−1DW
in anthers. Late-sown HT genotypes had significantly higher
sucrose concentrations in leaves (18.4–21.3 mg g−1DW) and
anthers (11.8–13.4 mg g−1DW) than sensitive genotypes (leaves
10.6–13.2 mg g−1DW; anthers 7.5–9.1 mg g−1DW).
The leaves and anthers of NS plants had 3.1–4.2 units
and 2.2–2.9 units of sucrose phosphate synthase (SPS) activity,
respectively (Figure 6), but the activity was significantly less in
LS plants (1.4–3.1 units in leaves; 1.1–2.2 units in anthers). SPS
activity was significantly higher in HT genotypes, especially in
the anthers, which had 50–75% more SPS activity than those of
HS genotypes. No significant differences were noticed among the
FIGURE 5 | (A) Ascorbate (ASC) and (B) reduced glutathione (GSH) in normal-sown (NS) and late-sown (LS) genotypes. Small vertical bars on histograms represent
standard errors. LSD (P<0.05; represented as small vertical line close to y-axis) for ASC (4.7) and GSH (4.9); T, tolerant; S, sensitive.
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Sita et al. Heat Stress and Lentil Genotypes
FIGURE 6 | (A) Sucrose phosphate synthase (SPS) and (B) sucrose (SUC) in leaves and anthers of normal-sown (NS) and late-sown (LS) lentil genotypes. Small
vertical bars represent standard errors. LSD (P<0.05; represented as small vertical line close to y-axis) for SPS (leaves: 0.43; anthers: 0.39) and SUC (leaves:1.9,
anthers: 1.7); T, tolerant; S, sensitive.
tolerant genotypes for sucrose concentration and SPS activity in
leaves and anthers.
Reproductive Traits
The flowers of LS plants showed significant damage to their
morphology due to heat stress (Figure 7). The structure of
anthers and pollen grains was adversely affected (Figures 8,9).
Late-sown plants had significantly less pollen viability
(41.7–66.9%) than NS plants (78.4–83.4%) (Table 8). Under
LS conditions, HT genotypes had more viable pollen (62.4–
70.3%) than HS genotypes (40.9–45.3%) when late-sown.
HT genotypes had 13.3–23.9% less pollen load compared
with 36.6–42% less in HS genotypes (Table 8). In vitro pollen
germination ranged from 70.3 to 78.2% in NS plants, but
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