ORIGINAL RESEARCH

published: 07 March 2019

doi: 10.3389/fpls.2019.00244

Stigma Functionality and Fertility Are

Reduced by Heat and Drought
Co-stress in Wheat
Attila Fábián, Eszter Sáfrán, Gabriella Szabó-Eitel, Beáta Barnabás and Katalin Jäger*
Plant Cell Biology Department, Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences,
Martonvásár, Hungary

As a consequence of climate change, unpredictable extremely hot and dry periods are
becoming more frequent during the early stages of reproductive development in wheat
(Triticum aestivum L.). Pollen sterility has long been known as a major determinant of
fertility loss under high temperature and water scarcity, but it will be demonstrated here
that this is not the exclusive cause and that damage to female reproductive organs also
contributes to losses of fertility and production. Changes in the phenology, morphology,
and anatomy of female reproductive cells and organs, in the ROS and RNS generation of
stigmatic papilla cells, and in fertility and yield components in response to simultaneous
high temperature and drought at gametogenesis were studied in two wheat genotypes
with contrasting stress responses. The combination of high temperature (32/24◦ C)
and total water withdrawal for 5 days at gametogenesis altered the phenology of
Edited by: the plants, reduced pollen viability, modified the morphology and the anatomy of the
Luisa M. Sandalio,
Spanish National Research Council
pistils, enhanced the generation of ROS and RNS, intensified lipid peroxidation and
(CSIC), Spain decreased the NO production of stigmatic papilla cells, all leading to reduced fertility
Reviewed by: and to production loss in the sensitive genotype, depending on the position of the floret
Delphine Laurinda Fleury,
on the spike. Reduced functionality of female and male reproductive parts accounted for
University of Adelaide, Australia
Irene Serrano, 34% and 66%, respectively, of the total generative cell- and organ-triggered fertility loss.
University of Göttingen, Germany
Keywords: anatomy, fertility, heat and drought co-stress, morphology, RNS, ROS, stigma, wheat
*Correspondence:
Katalin Jäger
[Link]@[Link]
INTRODUCTION
Specialty section:
This article was submitted to Wheat has a leading role in human nutrition and animal feed in the world with the largest
Plant Abiotic Stress, harvested area (220.1 million hectares) and the second largest production (749.5 million tons)
a section of the journal among cereals (FAOSTAT, 2016). With the continuously rising human population of the planet
Frontiers in Plant Science and the constant decline in agricultural land availability and quality, forecasted trends of yield
Received: 25 October 2018 increase will not be sufficient to satisfy future demand (Ray et al., 2013; Zandalinas et al., 2018).
Accepted: 13 February 2019 The enhancement of yield stability even under unfavorable environmental conditions is one of the
Published: 07 March 2019
primary goals of wheat breeders (Lamaoui et al., 2018). Among the extreme weather events, high
Citation: temperature and drought are expected to be the main yield decreasing factors (IPCC, 2014; Lesk
Fábián A, Sáfrán E, Szabó-Eitel G, et al., 2016). Globally, drought accounts for 21% yield loss on average (Daryanto et al., 2016). A 1◦ C
Barnabás B and Jäger K (2019)
increase in global temperature could reduce the global wheat yield by 4.1–6.4% depending on the
Stigma Functionality and Fertility Are
Reduced by Heat and Drought
method used for yield projection (Liu et al., 2016). It has been reported that more than 40% yield
Co-stress in Wheat. fluctuation of wheat can be attributed to climate change (heat waves and drought) at the global,
Front. Plant Sci. 10:244. national, and subnational scales (Zampieri et al., 2017). The growth, physiological, and metabolic
doi: 10.3389/fpls.2019.00244 responses of plants to a combination of heat and drought (HD) stresses are unique and cannot be

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

directly extrapolated from the responses to each of these information was given on the structural changes and processes
stresses separately (Rizhsky et al., 2002, 2004). Different stress underlying this phenomenon. However, the stigma, which
combinations should be handled as a new state of stress in plants, plays an essential role in reproductive processes, is the most
requiring novel types of defense and acclimation responses delicate but the least protected female organ, making it the most
(Suzuki et al., 2014). sensitive to adverse environmental conditions. If receptive, it
The sensitivity of a plant to environmental factors depends provides the exact conditions required for pollen germination
on the species, genotype and developmental stage, and on the and the sustained growth and guidance of the pollen tube
duration and severity of the stress. Heat and drought stress through the pistil and ovary (Heslop-Harrison, 2000), but no
during reproductive development may seriously affect crop yields information is available on the effect of HD co-stress on its
(Barnabás et al., 2008), which can be attributed especially to the anatomy and functionality.
high sensitivity to stress shown by pollen development (Saini Both extreme high temperatures and water shortage lead
and Aspinall, 1981; Saini et al., 1984; Lalonde et al., 1997; Jäger to the excessive generation of reactive oxygen species (ROS)
et al., 2008; for reviews, see Dolferus et al., 2011; De Storme and and reactive nitrogen species (RNS), which function as
Geelen, 2014). Compared to pollen dysfunction, the significance signal transduction molecules, but can also cause extensive
of the damage sustained by the physically better protected female cellular damage when the balance between the production and
reproductive cells and organs is generally considered as a minor scavenging of these compounds is impaired (Hasanuzzaman
factor in yield loss (Hedhly, 2011; Giorno et al., 2013); therefore et al., 2012; Choudhury et al., 2017; Zandalinas et al., 2018). ROS
less attention has been paid to the heat and drought sensitivity of and RNS are partially reduced or activated forms of molecular
their development. oxygen and nitrogen (del Río, 2015). Small amounts of these
Although there are emerging evidences of the sensitivity of radicals and compounds are produced continuously even under
female reproductive cell and organ development to heat or favorable conditions, particularly in the plastids, mitochondria,
drought stress per se in sorghum, rice, maize, wheat, tomato, peroxisomes, cytosol, and apoplast. The most important types
and canola (Saini and Aspinall, 1981; Saini et al., 1983; Mitchell of reactive radicals and compounds are singlet oxygen (1 O2 ),
and Petolino, 1988; Polowick and Sawhney, 1988; Jagadish et al., superoxide anion (O2 •− ), hydrogen peroxide (H2 O2 ), hydroxyl
2010; Prasad et al., 2011; Onyemaobi et al., 2017; Djanaguiraman radical (OH• ), nitric oxide (NO), and peroxynitrite (ONOO•− ;
et al., 2018; Pan et al., 2018), no information is available on the Molassiotis and Fotopoulos, 2011; Demidchik, 2015). These
combined effect of these two stresses. Majority of studies focus on molecules differ greatly in their lifespan, on a nanoseconds
the effect of heat or drought stress during meiosis and anthesis to seconds scale. ROS and RNS also show diverse reactivity,
and little attention has been paid to gametogenesis. During from moderate (O2 •− ) to very high (OH• , ONOO•− ; Waszczak
this process, if undisturbed, the sexual organs and gametes et al., 2018), being able to oxidize lipids, proteins, carbohydrates
complete their development, reach their final size and accumulate and nucleic acids, therefore effectively impairing the structural
the starch reserves needed for successful fertilization and the integrity of cells when present in large amounts (Vandelle
nourishment of the first cell division cycles of the embryo and and Delledonne, 2011; Demidchik, 2015). On the other hand,
the endosperm. the signaling role of ROS and RNS has been revealed in
Despite their central role in plant reproduction, the both developmental and stress reaction processes in the past
vulnerability of wheat pistils to heat or drought stress has decade (Waszczak et al., 2018). Although a certain amount of
hardly been investigated to date (Saini and Aspinall, 1981, information is available on the role of the ROS content of the
1982; Saini et al., 1983; Prasad and Djanaguiraman, 2014; stigma and stigmatic papillae in developmental changes and
Onyemaobi et al., 2017). Saini and Aspinall (1982) and Saini pollen incompatibility processes (McInnis et al., 2006; Serrano
et al. (1983) reported reduced fertility and altered ovary and et al., 2010, 2012, 2015; Domingos et al., 2015; Zafra et al.,
ovule development in 30% of wheat pistils as a consequence 2016), there are no data on the environmental stress-induced
of high temperatures during meiosis. Wheat plants, similarly ROS and RNS generation in this delicate and important organ.
to other Gramineae species, possess two-branched, feathery, As generative processes show significant vulnerability to heat
dry plumose type stigmas (Heslop-Harrison and Heslop- and drought stress, it can be hypothesized that ROS and RNS
Harrison, 1980; Heslop-Harrison, 1992). The stigma tissues play an important role in the reduction in fertility and in
have multiple tasks during pollination, all of which are crucial consequent yield loss.
for successful fertilization: the capture and hydration of the The sensitivity of female reproductive tissues to simultaneous
pollen, pollen tube guidance and transmission (Heslop-Harrison, heat and drought stress is not well understood. Addressing
1979). The first three of these four cardinal steps occur on the the morphological, anatomical, physiological, and molecular
receptive secondary branches of the stigma. In wheat, these mechanisms conferring sensitivity and tolerance to HD co-stress
branches are composed of four rows of highly vacuolated will help to develop wheat genotypes capable of adapting to
papilla cells, with a centrally located nucleus and a thin layer a changing climate. Hence, the objectives of this study were
of marginal cytoplasm (Heslop-Harrison and Heslop-Harrison, to (1) reveal the combined effect of heat and drought co-
1980). Although Prasad and Djanaguiraman (2014) found stress during gametogenesis on the morphology, structure and
that wheat stigmas and ovaries became desiccated following functionality of female reproductive cells and organs, and on
exposure to high temperature for 5 days before anthesis and the yield components of wheat genotypes with contrasting HD
that the pollen capturing ability of the stigma decreased, no tolerance; (2) shed light on the HD stress induced ROS and RNS

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

generation of stigmatic papilla cells, and (3) unravel the link

between HD stress-induced oxidative damage and fertility loss.

MATERIALS AND METHODS

Plant Material, Plant Cultivation, and
Stress Conditions
Two winter wheat genotypes, the drought-tolerant Plainsman
V1 and the drought-sensitive Cappelle Desprez2 were used in
the experiments. In previous studies (Jäger et al., 2008, 2014;
Fábián et al., 2011) significant differences were found in the
fertility loss induced by heat and drought stress in the varieties
Cappelle Desprez and Plainsman V, making them suitable for
examining the effect of HD co-stress on female reproductive cells
and organs. Plants (n = 50 per genotype and treatment) were
planted in pots containing 2 kg of a soil-sand-peat mixture ([Link],
v/v/v) after 7 weeks of vernalization at a temperature of 4◦ C,
and grown in growth chambers (Conviron, Winnipeg, Canada)
using the spring climatic program T1 (Tischner et al., 1997) under
optimum environmental conditions until the mid-uninucleate
stage of microspore development (hereinafter referred to as MU).
The max/min day/night temperature in the growth chambers
was 22/14◦ C, and irrigation was carried out regularly in the FIGURE 1 | Morphology of wheat reproductive organs and male reproductive
morning at a rate of 150 ml/day. The pots were randomly cells of the Cappelle Desprez winter wheat variety at (A,C) the
arranged and then rearranged every week to reduce border effects mid-uninucleate stage of microspore development and (B) anthesis. a, anther;
f, filament; l, lodicule; n, nucleus; o, ovary; p, palea; st, stigma; asterisk,
and minimize any variation in light and temperature. The fact
aperture. Bar represents (A,B) 1 mm; (C) 25 µm.
that the microspores were in the MU stage was checked by
acetocarmine staining followed by light microscopy (Figure 1)
and based on the distance between the auricles of the flag
Devices Ltd., Cambridge, United Kingdom) on the basis of the
leaf and the penultimate leaf. Plants with main spikes at MU
changes recorded in the apparent dielectric constant at full water
were identified each morning, tagged and transferred to control
saturation and during the treatments. The relative water content
or stress chambers. HD stress was generated by total water
(RWC) of flag leaves excised from the main tillers of 15 plants per
withholding at 32/24◦ C max/min temperature for 5 days from
genotype and treatment was determined at the end of HD stress
MU until flowering under controlled conditions. During this
using the fresh weight (FW) at excision, the saturated weight
period the mean volumetric water content of the soil dropped
(SW) after 24 h re-hydration in distilled water at 25◦ C in the
from 37.2 to 5.1% that was equal to 13 kPa and 10,549 kPa
dark, and the dry weight (DW) after oven drying for 24 h at
soil water potential, respectively. The relative daily max/min air
80◦ C. The leaf RWC was calculated using the following equation:
humidity was 75%/65% and 65%/30% in the control and stress
RWC(%) = FW−DW
SW−DW × 100.
chambers, respectively. The light intensity during the experiment
was 300 µmol m−2 s−1 . At 10 am on the day of flowering
(at the end of the 5-day treatment) samples were collected
Phenology
and 20 plants of each genotype and treatment were re-irrigated The days from MU to anthesis and physiological maturity were
and grown to full maturity under control conditions at a final noted for each genotype. MU was determined as described above.
max/min temperature of 32/24◦ C. If not otherwise indicated, all A plant reached anthesis when the first anther appeared on
the below-mentioned measurements and samplings were carried the middle of the isolated and tagged main spike. Physiological
out in at least three biological and three technical repetitions (per maturity was reached when the peduncle of the main spike
genotype and treatment) at anthesis. The effect of treatments was became yellow.
assessed for the tagged main tillers only.
Determination of Yield Components
Measurement of Water Content The main tillers of control and drought-stressed plants (n = 20
The volumetric water content of the soil (n = 10 pots per per genotype and treatment) were hand-harvested and the
genotype and treatment) was monitored using an HH2 moisture spikes were threshed at full maturity. The plant height and
meter connected to an SM200 soil water sensor (Delta-T the spikelet number, grain number and grain weight of both
the basal and upper spike halves were determined and mean
1
[Link] values were calculated for each treatment and cultivar. The
2
[Link] thousand-grain weight (TGW) was calculated from the data.

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

The fertility index was calculated as the quotient between the Arnhem, Netherlands). The area of the ovaries and the length
potential and actual grain number per spikelet. Three grains per of the stigmas was measured using Image-Pro Plus 7.0 software
a spikelet characteristic of both genotypes were considered when (Media Cybernetics, Rockville, MD, United States).
potential grain number of intact spikelets was determined. In case
of truncated or manipulated spikes, two grains per spikelet were Light Microscope Studies: Pistil Anatomy
considered when potential grain number was calculated. Stigmas (n = 4 per genotype and treatment) from the central
regions of both the lower and upper halves of the spikes were
Pollination Experiment excised prior to anthesis, incubated in Tris-HCl buffer (10 mM,
In order to shed light on the contribution of HD stress-induced pH 7.4) containing 5 µM Syto-63 fluorescent nucleic acid probe
stigma injury to fertility reduction in Cappelle Desprez, 90 plants (Thermo Fisher Scientific) in the dark for 10 min at room
were grown until MU under the control conditions described temperature and washed for 3 min in Tris-HCl buffer. As Syto-63
above. At MU the plants were divided into two groups. While stains the nucleus and the cytoplasm with different intensities it
the plants in the 1st control group were grown further under was used as a general stain for the visualization of the stigma
optimum conditions, plants of the second group were subjected papilla cell structure. Fluorescence was detected using a Leica SP8
to HD co-stress until flowering. Only the main tillers of mother confocal laser scanning microscope (Leica Microsystems GmbH,
plants were used in this experiment. The control group consisted Wetzlar, Germany). Syto-63 was excited at 633 nm and the
of four sub-groups: (i) pollen donor plants (n = 30), half of which emitted fluorescence was detected at 650–700 nm. In addition,
were planted 2 weeks earlier in order to compensate for the differential interference contrast (DIC) images of the papilla
more rapid development of HD-stressed plants, (ii) plants with cells were collected during confocal image acquisition using a
intact florets for free pollination (n = 15), (iii) pollen recipient transmitted light detector.
plants in which the top third of the glumes, paleas and lemmas For histological studies, pistils (n = 4 per floret position,
in all the primary and secondary florets were cut off and the genotype and treatment) isolated from central regions of both
central florets were removed 3 days before anthesis (truncated the lower and upper halves of the spikes were collected just
florets; DBA; n = 15) in order to simulate the majority of the before anthesis, fixed in 50 mM Na-cacodylate buffer (pH 7.2)
damage sustained by the florets during emasculation, and (iv) containing 2.5% glutaraldehyde (v/v) and 4% formaldehyde (w/v)
pollen recipient plants treated as in (iii) and also emasculated 3 overnight at 4◦ C, washed, dehydrated in an ethanol series and
DBA (manipulated florets; n = 30). The treated group consisted gradually infiltrated with LR white acrylic resin (Ted Pella,
only of groups ii, iii, and iv. Care was taken not to cut or Redding, CA, United States) according to the manufacturer’s
touch the stigmas during these manipulations. All the spikes were instructions. The resin was polymerized under UV light at
bagged in order to prevent pollination by neighboring ones and −20◦ C. Semi-thin sections (1 µm) were serially sectioned at
the date of floret manipulation was recorded on crossing tags. the sagittal plane of the ovaries and at the transverse plane
At the onset of anthesis (dehiscent anthers visible in similarly of the stylodia using an Ultracut-E microtome (Reichert-Jung,
developed spikes in group ii) both the control and HD-stressed Heidelberg, Germany) and were stained with periodic acid-Schiff
main spikes with mutilated and emasculated florets (group iv) (PAS) and 1% Amido Black for polysaccharides and proteins,
were hand-pollinated with pollen from the pollen donor control respectively. Stained sections were mounted in 50% glycerol
plants (group i) and were grown to maturity under optimum containing 7% acetic acid, examined under a BX51 light
conditions. The fertility index of both sub-regions (base, top) of microscope (Olympus, Tokyo Japan) and analyzed using an
the main spikes was calculated and analyzed. Image-Pro Plus 5.1 image analysis software (Media Cybernetics,
Inc., Bethesda, MD, United States).
Pollen Viability Assay
Wheat pollen collected separately from the base and top of the
spikes was incubated in 0.1 M pH 7.2 Sorensen’s phosphate buffer
Detection of Reactive Oxygen Species,
containing 0.5% TTC for 15 min in the dark at 37◦ C. In this assay, Reactive Nitrogen Species, and Lipid
the TTC is converted into red formazan dye by dehydrogenases if Peroxidation in Stigmatic Papilla Cells
the pollen cells are viable. Pollen viability was calculated as the The ROS and RNS contents and the quantity of lipid peroxidation
percentage of dark red pollen to the total number of counted products were determined in the stigmas (n = 5 per genotype and
pollen grains across 20 microscopic field views, such that at least treatment) isolated from the central regions of both the lower and
2,800 pollen grains were assessed per genotype and treatment. upper halves of six main spikes. Samples were collected just prior
to anthesis. Pistils with intact stigmas were incubated in Tris-HCl
Morphometric Analysis of the Pistils buffer (10 mM, pH 7.4; except labeling with C11-BODIPY where
The pistils (n = 8 in Plainsman V; n = 10 in Cappelle Desprez) 60 mM Sorensen’s phosphate buffer pH 7.4 was used) containing
in one primary floret in each spikelet, located along one side of the relevant fluorescent probe in the dark, followed by washing
the rachis and numbered starting at the bottom, were collected three times for 3 min in Tris-HCl buffer. The stigmas were
from three plants per genotype and treatment at anthesis. carefully excised from the ovaries and the fluorescent signal
The rudimentary florets located both at the base and top of was visualized immediately using a Leica SP8 laser scanning
the spikes were not taken into account. Micrographs were confocal microscope (Leica Microsystems GmbH, Wetzlar,
taken using a DiscoveryV8 stereomicroscope (Zeiss, Darmstadt, Germany). General cellular oxidative stress was assessed using
Germany) equipped with an HD Ultra camera (Euromex, 20 ,70 -dichlorodihydrofluorescein diacetate (H2 DCFDA, Sigma) at

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

10 µM final concentration at 37◦ C for 30 min, with excitation at values were corrected by a factor of cell wall autofluorescence
488 nm and signal detection at 517–527 nm. Mitochondrial O2 •− determined during our preliminary experiments on unstained
was detected using MitoSOX Red dye (Thermo Fisher Scientific) control and HD treated papilla cells. The ratio of oxidized
at 5 µM final concentration at room temperature (RT) for 30 min, BODIPYTM 581/591 C11 probes was calculated from pixel
with excitation at 514 nm and signal detection at 580–700 nm. intensities measured in ROIs using the following equation
The superoxide anion radical (O2 •− ) content in the cytosol was (Pap et al., 2000):
labeled with dihydroethidium (DHE, Sigma) at 10 µM final
Intensityoxidized(500−560nm)
concentration at 37◦ C for 30 min, with excitation at 514 nm %oxidized = ∗ 100
and signal detection at 580 to 700 nm. Hydroxyl radical (OH• ) Intensityoxidized(500−560nm)
and peroxynitrite (ONOO− ) were monitored using aminophenyl + Intensitynon−oxidized(570−620nm)
fluorescein (APF, Sigma) at 10 µM final concentration at RT
for 30 min, with excitation at 488 nm and signal detection Statistical Analysis
at 500–550 nm. Extracellular hydrogen peroxide (H2 O2 ) was All measurements were carried out in at least three biological
detected using Ampliflu Red: samples were incubated with and three technical repetitions. Data were subjected to ANOVA
Ampliflu Red and horseradish peroxidase at 50 µM and 0.2 U/ml (SPSS version 16.0, IBM Corp., Armonk, NY, United States). The
final concentration, respectively, at RT for 30 min, with excitation mean values were compared by the Tukey’s multiple range test
at 561 nm and signal detection at 565–650 nm. Intracellular H2 O2 taking P ≤ 0.05 as significant to compare the differences between
was visualized with dihydrorhodamine 123 (DHR 123, Thermo treatments and genotypes. Pearson’s correlation coefficient was
Fisher Scientific) at 5 µM final concentration at RT for 30 min, used to identify relationships between the measured characters.
with excitation at 514 nm and signal detection at 520–600 nm. Mean values along with standard deviations are presented in the
Nitric oxide (NO) was labeled with 4-amino-5-methylamino- tables and figures.
20 ,70 -difluorofluorescein diacetate (DAF FM-DA, Thermo Fisher
Scientific) at 10 µM final concentration at RT for 60 min, with
excitation at 488 nm and signal detection at 500–580 nm. Lipid RESULTS
peroxidation was monitored using C11-BODIPYTM 581/591
(Thermo Fisher Scientific) at 1 µM final concentration at 37◦ C Reduced RWC of Wheat Plants After HD
for 30 min. The fluorescent signals of the non-oxidized and Stress
oxidized forms were acquired using simultaneous excitation. The Combined heat and drought stress applied for 5 days prior to
non-oxidized form of the dye was excited at 561 nm and signals anthesis induced a substantial reduction in the RWC of the flag
were detected at 570–620 nm. The oxidized form of the probe was leaves in both genotypes, but this reduction in Cappelle Desprez
excited at 488 nm, signals were detected at 500 to 560 nm. The RWC (43%) was significantly (P ≤ 0.05) more pronounced
dimer formation of C11-BODIPYTM molecules with red-shifted than in Plainsman V (21%; Table 1). The leaves of treated
fluorescence (excimers; Pap et al., 1999) was monitored by Cappelle Desprez plants showed increasing visible symptoms of
fluorescence measurement at 570–630 nm after excitation at dehydration, starting around noon on the third day of treatment,
488 nm. ROS- and RNS-sensitive fluorescent dyes were used while leaf rolling was only observed on the flag leaves of treated
simultaneously with ROS scavengers as negative controls and Plainsman V plants on the fifth day of treatment.
ROS generating treatments as positive controls for the proper
evaluation of the fluorescence intensities detected in control and Simultaneous Heat and Drought
HD treated. Specific localization patterns of the used probes were
evaluated during the preliminary experiments (Supplementary
Reduced Fertility and Production
The main spikes of the Cappelle Desprez variety were
Table 1) according to the literature (Ortega-Villasante et al.,
significantly (P ≤ 0.05) longer (10.7 ± 0.1 cm) than those
2016). All microscope settings (i.e., laser power, detector gain,
of Plainsman V (9.2 ± 0.4 cm), and consisted of 25.4 ± 1.7
detection spectra, etc.) were saved for each fluorescent probe and
and 17.9 ± 1.2 spikelets, respectively. No significant HD-
kept the same throughout all the repetitions.
stress dependent reduction in spike length was observed
Quantification of Fluorescent Signals
Fluorescent signal intensities were measured on the images
TABLE 1 | Relative water content of control and HD-stressed Cappelle Desprez
using Leica Advanced Fluorescence software v3.1.5.1638 and Plainsman V flag leaves at anthesis.
(Leica Microsystems GmbH, Wetzlar, Germany). Relative
fluorescence intensities were measured on 60 micrographs per Genotype Flag leaf water content (%)
genotype, treatment and fluorescent probe, using 10 regions of
Control HD stress
interest (ROIs) per micrograph containing only the organelles
emitting specific signals (e.g., mitochondrion, cell wall, vacuole). Plainsman V 88.61 ± 4.27a 70.03 ± 4.94b
Unspecific autofluorescence originating from cell walls was Cappelle Desprez 87.58 ± 1.13a 50.12 ± 1.38c
not taken into account during quantification. The measured
Values represent means ± standard deviations. Means with different superscripts
data were normalized against the background fluorescence. are significantly different at least at the P ≤ 0.05 level of probability. HD,
In the case of Ampliflu Red, measured fluorescence intensity simultaneous heat and drought.

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

in either of the genotypes. Irrespective of the genotype pollination experiment was conducted on Cappelle Desprez
and treatment, the grain number and fertility of florets plants, where the effect of floret position, floret truncation
developing in the upper part of the spikes were significantly and manipulation was also considered. Floret location (top or
lower than the relevant parameters of the basal florets. base of the spike) had a significant effect on fertility in both
Although combined stress had no effect on the fertility control and HD-stressed Cappelle Desprez plants (Table 3). The
ratio of Plainsman V, the fertility of HD-stressed Cappelle cutting off one-third of the bracts (glumes, paleas, lemmas)
Desprez florets located in the base and top regions showed in the primary florets and the removal of the central florets
a significant decrease of 39% and 56%, respectively. Slight, (truncation, group iii) from the control spikelets caused a
but not significant HD stress-induced decreases and increases position-independent loss in fertility (base: 21%, top: 25%).
in TGW occurred in the basal and top florets, respectively, Compared to the truncated and free-pollinated control florets
of both genotypes. HD stress only induced a non-significant (group iii), the hand pollination of truncated and emasculated
8% loss in plant production of Plainsman V. In contrast, (manipulated, group iv) control florets with control pollen did
as a consequence of the loss in fertility, the production of not reduce fertility. Compared to the free-pollinated intact
Cappelle Desprez was severely reduced by 55% (Table 2). control florets (group ii), HD stress induced a significant 62%
According to the yield components recorded after co-stress and 33% loss in the fertility of truncated free-pollinated (group
during microgametogenesis, Plainsman V and Cappelle iii) and manipulated and hand-pollinated (group iv) florets,
Desprez were considered as HD-tolerant and HD-sensitive respectively (Table 3). The removal of one-third of the bracts
varieties, respectively. had a similar fertility-reducing effect in HD-treated florets (base:
24%; top: 26%) as in the control. In free-pollinated HD-stressed
spikes, there was a significant difference between the fertility
Floret Position, Truncation of Bracts, and loss in florets located in the base (45%) and top (56%). Both
HD-Triggered Stigma Dysfunction pistil- and-pollen dependent fertility loss varied significantly
Influenced Fertility with position, being 22% and 36% in florets located in the
In order to assess the effect of the stigma dysfunction triggered upper halves of the spikes and 15% and 32% in basal florets,
by HD stress on fertility loss in the sensitive genotype, a respectively (Table 3).

TABLE 2 | Changes in yield components of the main spikes after HD stress in the winter wheat varieties Plainsman V and Cappelle Desprez.

Yield Spike half

components

Plainsman V Cappelle Desprez

Control HD stress Control HD stress

Base Top Base Top Base Top Base Top

Spikelet no. 9.5 ± 0.4b 8.7 ± 1.0b 9.3 ± 0.3b 8.5 ± 0.6b 13.3 ± 1.3a 12.1 ± 0.7a 12.7 ± 0.7a 12.1 ± 0.9a
Grain no. 17.4 ± 1.8c 14.0 ± 2.6de 16.9 ± 1.0cd 14.2 ± 0.9e 27.4 ± 0.7a 20.7 ± 1.4b 15.4 ± 1.1e 8.9 ± 1.6f
Fertility (%) 61.4 ± 6.9a 53.8 ± 6.2bc 60.8 ± 2.0ab 50.6 ± 1.5c 66.8 ± 3.7a 55.8 ± 4.2bc 40.7 ± 0.5d 24.4 ± 2.6e
TGW (g) 42.3 ± 6.5a 38.0 ± 5.7a 37.7 ± 5.3a 39.0 ± 7.0a 40.1 ± 5.0a 31.3 ± 5.4a 27.3 ± 1.6a 33.5 ± 2.9a
Production (g) 0.73 ± 0.03b 0.52 ± 0.05cd 0.64 ± 0.12b 0.50 ± 0.08d 1.10 ± 0.15a 0.65 ± 0.13bc 0.42 ± 0.12de 0.30 ± 0.06e

Values represent means ± standard deviations. In each row, means with different superscripts are significantly different at least at the P ≤ 0.05 level of probability. Base,
lower half of the spike; HD, simultaneous heat and drought; Top, upper half of the spike; TGW, thousand-grain weight.

TABLE 3 | Effect of floret position, floret manipulation, and hand pollination with control pollen on fertility rates of the Cappelle Desprez variety.

Fertility % of the spikes with florets

Intact free Truncated and Manipulated and

pollinated (ii) free pollinated (iii) hand pollinated (iv)

Base Top Base Top Base Top

Control 75.6 ± 3.9a 61.6 ± 0.2b 59.9 ± 3.3bc 48.5 ± 0.3d 61.1 ± 1.0b 51.7 ± 0.2c
HD stress 41.4 ± 2.4e 27.4 ± 2.4fg 31.6 ± 0.3f 20.5 ± 0.1g 52.0 ± 0.5c 40.5 ± 2.7e

Values represent means ± standard deviations. Means with different superscripts are significantly different at least at the P ≤ 0.05 level of probability. Base, lower half
of the spike; HD, simultaneous heat and drought; top, upper half of the spike; truncated florets, the top third of the glumes, paleas and lemmas in all the primary and
secondary florets were cut off and the central florets were removed 3 days before anthesis; manipulated florets, truncated and emasculated 3 days before anthesis;
emasculation, removal of stamens.

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

Changes in Phenology Due to Combined

Stress
The adverse environmental conditions significantly shortened
the duration of microgametogenesis, which lasted for 7
days under optimum conditions. Independently of the
genotype, HD-stressed plants started flowering 3 days earlier.
Moreover, HD stress shortened the duration of grain filling
in both Plainsman V and Cappelle Desprez, by 10 and
14 days, respectively.

Effect of Co-stress on Spike and Anther

Morphology and Pollen Viability
Compared to their respective controls (Figures 2A,C), there
was no change in spike morphology in Plainsman V a week FIGURE 3 | Viability of control and HD-stressed Plainsman V and Cappelle
Desprez pollen in anthers located in the basal and top half of the spikes.
after HD treatment (Figure 2B), while the bracts of the apical
Columns represent means; vertical bars denote ± standard deviations.
florets in treated Cappelle Desprez spikes turned yellow and Different letters above columns indicate significant difference between means
the spikes showed strongly reduced fertility (Figure 2D and at least at the P ≤ 0.05 level of probability.
Tables 2, 3). The size of the anthers did not vary significantly
with the treatment.
The mean viability of Plainsman V pollen (80.4% ± 2.7) reduction varied with floret position, averaging 35% at the base,
was independent of the treatment or floret position. In contrast, 12% in the center and 31% at the top of the spikes. Some of the
for the genotype Cappelle Desprez, lower pollen viability was HD stressed Cappelle Desprez stylodia were shriveled, with fewer
observed in superior spikelets than inferiors under both optimum secondary branches (Figure 4).
conditions and heat and drought co-stress. Compared to the Compared to the control, no HD stress-induced structural
base (74.6% ± 4.3) the viability of control pollen located alterations were observed in Plainsman V stigmatic papilla cells.
in the top anthers was significantly (26%) lower. HD stress In contrast, as a consequence of HD stress, the papilla cells
had a severe effect on Cappelle Desprez pollen; compared of Cappelle Desprez partially lost their turgor and thus the
to their respective controls, viability of HD stressed pollen secondary stigma branches were shriveled. Light microscopic
cells decreased by 63% and 81% in basal and apical anthers, studies using DIC and Syto 63 staining revealed the structural
respectively (Figure 3). effects of HD treatment on the stigmatic papillae (Figure 5).
Untreated stigmatic papilla cells possessed disk-shaped nuclei
Morphological and Anatomical Changes wedged tightly in between the large vacuoles that occupied
Induced by HD Stress in Pistils almost the whole of the cell (Figures 5A–C). The cytoplasm was
Although neither the morphology (Figure 4), nor the size generally located on the periphery of the papilla cells, although
(data not shown) of the ovaries changed with the treatment, large cytoplasmic segment was also found in control outwardly
compared to their respective controls (Figures 4A,C), the length curved apical papilla cells. The stigmatic papilla cells of Cappelle
of HD-stressed stylodia (Figures 4B,D) was significantly reduced Desprez pistils, especially those located in the top half of the
in both genotypes (Table 4). The extent of genotype-independent spikes, showed signs of injury after treatment (Figures 5D–F).
The nuclei were reshaped and relocated from their original
position. Syto 63 staining revealed fragmentation of the nuclei
and cytoplasm (Figures 5D–F).
The transversally cut surface of both control and HD-stressed
stylodia was somewhat ovate at a quarter of the way from
the top, and consisted of large, vacuolated cortical cells (co)
surrounding a few small, cytoplasm-rich transmitting cells (tt).
Although HD stress had no effect on the structure of Plainsman
V stylodia, whether isolated from the top or base of the spike,
those in the upper part of Cappelle Desprez spikes were slightly
dehydrated. Halfway down the spike, control stylodia and those
of HD-stressed Plainsman V had a shield-like shape (Figure 6A).
A massive vascular bundle (vb) surrounded by cortical cells was
located on the lateral side of the stylodia, the medial side consisted
FIGURE 2 | Spikes of (A,C) control and (B,D) HD-stressed (A,B) Plainsman V, of small, turgid, well-demarcated transmitting cells with round
and (C,D) Cappelle Desprez varieties 7 days after anthesis (DAA). (D) Note
nuclei. Multiple layers of large, vacuolated cortical cells connected
sterile florets located in the upper half of HD-stressed Cappelle Desprez spike.
Bar represents 10 mm.
the two sides of the stylodium (Figure 6A). By contrast, as a
consequence of the HD stress-induced extensive degeneration of

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

these organs. The style (s), protruded into the ovary consisted
of 10–15 rows of vacuolated spindle-like cells with large ovate
nuclei. At the base the style took the form of a funnel that
ended between the tube cell (tc) layer and the outer layer of
the outer integument (oii; Figure 6D). The ovaries were well
developed; the mesocarp cells (me) accumulated a large amount
of starch (Figures 6D,E). The cross and tube cell layers were
discontinuous at the base of the two styles, where the cells of
the style were loosely connected to the outer layer of the outer
integument (Figure 6D). Chloroplasts were visible in the dense
cytoplasm of both the cross and tube cell layers (Figure 6F), but
chloroplasts accumulating starch deposits were only visible in a
few cell rows lining the base of the style. The ovary contained
a single embryo sac surrounded by integuments (Figures 6E,F).
The double layer of the outer integument was highly vacuolated
(Figure 6F). The two layers of the inner integument formed
the micropyle (mi) at the base of the ovule (Figures 6E,G).
The ovules were lined with the nucellar epidermis (ne) and
nucellus (n; Figure 6G). However, none of these were present in
the vicinity of the micropyle, while large quantities of nucellar
cells were observable at the chalaza (ch; Figure 6E). Irrespective
of the treatment, degraded nucellar cells (dn) were found in
the proximity of vacuolated antipodal cells (a; Figure 6H). The
egg apparatus consisted of two highly vacuolated synergids (sy)
with well-developed filiform apparatus (f), and the egg cell (e;
Figures 6G,I). Regardless of whether the two polar nuclei of the
central cell (c) were adjacent to the egg cell or not, they were
attached to it by means of cytoplasmic bridges (Figure 6G). Both
the egg cell and the central cell accumulated starch (Figure 6I).

Influence of HD Stress on ROS and RNS

Generation in the Stigma
The ROS and RNS production of control and HD stressed
stigmatic papilla cells was analyzed in both genotypes. In
order to estimate the general oxidative stress manifested in the
cells, non-fluorescent H2 DCFDA, an indicator of total ROS
was added to control and HD-stressed stigmas. H2 DCFDA,
after cleavage by oxidation was converted to the highly
fluorescent 20 ,70 -dichlorofluorescein. The fluorescence of the
oxidized H2 DCFDA showed vacuolar localization (Figure 7A).
FIGURE 4 | Morphology of female reproductive organs (pistils) dissected from Irrespective of the genotype a weak fluorescent signal was
(A,C) control and (B,D) HD-stressed (A,B) Plainsman V and (C,D) Cappelle
Desprez florets at anthesis (representative images). Note that control stigmas
observed in all the control stigmas and in those located in
are long with numerous secondary stigma branches, while HD-stressed thin the lower half of HD-stressed spikes. In contrast, significantly
stigmas are shorter with dehydrated, withered secondary branches typical of higher fluorescence was detected in HD-stressed papilla cells
pistils located in the top of the spike. Bar represents 5 mm. dissected from the upper florets of both varieties: a more
than two- and ten-fold significant (P ≤ 0.05) increase was
observed in Plainsman V and Cappelle Desprez stigmas,
almost all the cortical cells and part of the transmitting tissue, a respectively (Figure 7B).
mass of crushed cells was visible in HD-stressed stylodia isolated Mitochondrial O2 •− production was assessed by MitoSOX
from the top half of the sensitive genotype (Figure 6B), and as a Red staining. The specific signal of the oxidized fluorophore
consequence of the damage these turned into dumbbell-shaped was detected in mitochondria located in the narrow, peripheral
bilobed structures (Figure 6C). A few intact cortical cells were cytoplasm of the papilla cells (Figure 7C). There was no
visible exclusively in the proximity of the vascular bundle. significant difference between the control and HD-treated
Irrespective of the genotype and treatment, unilocular ovaries stigmas in either spike halves of Plainsman V in terms
and ovules showed similar anatomy in both control and of mitochondrial superoxide accumulation. In contrast,
HD-treated florets. No signs of dehydration were observed in mitochondrial O2 •− generation was significantly increased in

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TABLE 4 | Stigma length in control and HD-stressed Plainsman V and Cappelle Desprez pistils at anthesis.

Pistil position Stigma length (mm)

Plainsman V Cappelle Desprez

Control HD stress Control HD stress

1.92 ± 0.22i 1.25 ± 0.17k

2.44 ± 0.44gh 1.62 ± 0.17j
2.81 ± 0.26g 2.05 ± 0.22hi 2.83 ± 0.13g 2.15 ± 0.45hi
3.33 ± 0.38cdef 2.74 ± 0.22gh 3.02 ± 0.34efg 2.36 ± 0.27h
3.60 ± 0.31bc 3.08 ± 0.19efg 3.19 ± 0.10ef 2.78 ± 032g
3.76 ± 0.19a 3.40 ± 0.17cde 3.50 ± 0.20bcd 3.03 ± 0.39efg
3.74 ± 0.17ab 3.34 ± 0.30cdef 3.31 ± 0.24cdef 2.76 ± 0.32g
3.43 ± 0.36cde 2.95 ± 0.23fg 2.80 ± 0.16g 2.36 ± 0.15h
3.44 ± 0.36bcde 2.94 ± 0.13fg 2.57 ± 0.13gh 1.68 ± 0.15j
2.26 ± 0.32def 2.14 ± 0.14hi 1.76 ± 0.24j 1.13 ± 0.15k

Values represent means ± standard deviations. Means with different superscripts are significantly different at least at the P ≤ 0.05 level of probability. HD, simultaneous
heat and drought.

the upper stigmas of treated Cappelle Desprez (Figure 7D). emitting superoxide-dependent fluorescence were also detected,
Relative amount of the cytoplasmic superoxide radical was presumably due to the incorporation of oxidized DHE into
measured using DHE, which was detected in the nucleus after mitochondrial DNA (Figure 7E). Irrespective of the genotype
oxidation in the cytosol. Although the fluorescent signal was and floret position (Figure 7F), the generation of cytoplasmic
observed mainly in the nucleus, small cytoplasmic objects O2 •− was significantly increased as a consequence of HD stress.

FIGURE 5 | (A–C) Control and (D–F) HD-stressed stigmatic papilla cells of the stress-sensitive Cappelle Desprez variety at anthesis. Images were taken both in
(A,D) differential interference contrast (DIC) and (B,E) fluorescent mode after staining with a SYTO-63 nucleic acid probe. Merged images are shown in (C,F).
Arrows, marginally located cytoplasm; arrowhead, nucleus; v, vacuole. Note fragmented nuclei in HD-stressed stigmatic papilla cells (D–F). Bar represents 20 µm.

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

FIGURE 6 | Micromorphology of non-fertilized Cappelle Desprez stylodia and ovules. (A) Cross-sectioned shield-like control stylodium halfway along the entire
length. (B) Crushed mass of degenerated cortical cells in HD-stressed stylodia. (C) Structure of a HD-stressed bilobed stylodium. (D) Transmitting tissue surrounded
by the mesocarp, with the ovule beneath it. (E) Egg cell, central cell, and antipodals located in a sagittally sectioned ovule lined with a position-dependent amount of
nucellar cell layers. (F) Cell layers encompassing the ovule. (G) Egg cell apparatus located at the mycropilar end of the ovule, joined to the central cell with
cytoplasmic bridges. (H) Degrading cell layers of the nucellus associated with antipodal cells. (I) Egg cell apparatus consisting of the egg cell and the synergids. a,
antipodals; c, central cell; cc, cross cell; ch, chalaza; co, cortical cells; cco, crushed cortical cells; dn, degraded nucellar cells; e, egg cell; ep, epidermis; f, filiform
apparatus; iii, inner layer of the inner integument; ioi, inner layer of the outer integument; ls, lateral side of the stylodium; me, mesocarp; mi, micropyle; ms, medial
side of the stylodium; n, nucellus; ne, nucellar epidermis; oii, outer layer of the inner integument; ooi, outer layer of the outer integument; s, stylodium; sp, stigmatic
papilla cells; sy, synergid; tc, tube cell; tt, transmitting tissue; vb, vascular bundle; pink coloration, carbohydrates; blue coloration, proteins. Scale bar represents
(A–D,H) 100 µm; (F,G) 50 µm; (I) 20 µm.

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

FIGURE 7 | Localization of (A,C,E,G) fluorochromes and (B,D,F,H) relative fluorescence of stigmatic papillae cells observed after labeling with (A,B) H2 DCFDA,
(C,D) MitoSOX Red, (E,F) DHE and (G,H) APF in stigma papilla cells of control and HD-stressed Plainsman V and Cappelle Desprez wheat plants at anthesis.
Specificity of used ROS probes: H2 DCFDA, total ROS; MitoSOX Red, mitochondrial O2 - ; DHE, cytosolic O2 - ; APF, intracellular highly reactive OH and ONOO- .
Arrows, specific MitoSox Red signal from the mitochondria; arrowheads, non-specific autofluorescence from the cell wall. In each histogram, letters above columns
indicate significant differences between means at the P ≤ 0.05 level of probability. Bar represents (A,C,E,G) 10 µm and (C inset) 1 µm.

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

The amount of highly ROS was estimated by APF labeling, positive correlation (r = 0.55) was found between the fertility ratio
which allows the detection of hydroxyl radical (OH• ) and and intracellular NO content of the papilla cells.
peroxynitrite (ONOO− ). Relatively less radical-specific
fluorescence was observed in the case of APF if compared
to other probes used in this study (Figure 7G). In Plainsman DISCUSSION
V stigmas, no significant elevation of the fluorescent signal was
measured after HD treatment. In contrast, a strong, significant High temperature and drought often occur simultaneously
rise in specific fluorescence was measured in the top halves of during plant development causing severe yield loss in most
Cappelle Desprez spikes (Figure 7H). wheat-growing areas. Structural and functional anomalies
The relative content of extracellular hydrogen peroxide was occurring as a consequence of environmental stress during
measured using Ampliflu Red. A specific fluorescent signal reproductive processes have a serious influence on the success of
of resorufin, the product of the reaction between H2 O2 and fertilization and thus on yield production.
Ampliflu Red was detected in the apoplast (Figure 8A). HD stress As in many members of the Poaceae family, the stigma
had no effect on the apoplastic hydrogen peroxide content of of wheat is dry and plumose (Figure 1B) and the pistil is
Plainsman V papilla cells, while co-stress induced a significant bifurcated. The primary branches, known as stylodia, are densely
increase in apoplastic resorufin fluorescence at both the base and covered with multiseriate secondary branches consisting of
top of Cappelle Desprez spikes (Figure 8B). The intracellular papilla cells. While no signs of dehydration or anatomical
generation of H2 O2 and peroxynitrite was monitored with anomalies were observed in the pistils of the tolerant genotype,
dihydrorhodamine 123, which after oxidation to rhodamine 123 moderately dehydrated secondary stigmatic branches were
(RH) accumulates in the mitochondrial membranes (Figure 8C), typical of HD-stressed Cappelle Desprez pistils isolated from the
reflecting the H2 O2 and peroxynitrite content of the cell. The upper half of the spikes. A similar phenomenon was observed
RH fluorescence did not change significantly in Plainsman V in wheat and sorghum stigmas when exposed to heat stress
papilla cells, while it showed a nearby threefold increase in per se (Prasad and Djanaguiraman, 2014; Djanaguiraman et al.,
stigmas located in the upper half of HD-treated Cappelle Desprez 2018). Moreover, the nuclei of stigmatic papilla cells changed
spikes (Figure 8D). their position and the nuclei and cytoplasm were fragmented.
The relative content of nitric oxide (NO) was detected using a The stylodia of the sensitive genotype were malformed, as the
DAF-FMDA probe. NO was shown to be localized in the vacuoles majority of the cortical cells and some of the transmitting cells
of stigmatic papilla cells (Figure 8E). Compared to the control, were crushed (Figure 6). No such environmental stress-induced
HD stress had no effect on the nitric oxide accumulation in structural anomalies have been described in angiosperms so far.
Plainsman V. However, a significant drop in the NO content of A genotype-independent reduction in the stylodium length was
Cappelle Desprez papilla cells was observed, irrespective of the detected in both HD-sensitive and -tolerant wheat genotypes
position of the florets in the spike (Figure 8F). after HD treatment, probably as a consequence of the arrested
cell enlargement induced by a significant decrease in plant RWC.
HD Stress Induced the Peroxidation of This contrasts with the findings of Jagadish et al. (2010) and Pan
et al. (2018), who reported unaffected stigma length and stigma
Membrane Lipids
exsertion, respectively, after high temperature stress.
Lipid peroxidation was evaluated using a C11-BODIPYTM
Heat and drought co-stress had no effect on the ovule
probe, which showed a specific staining pattern localized in the
or female gametophyte development in either of the wheat
membranes of papilla cells (Figure 8G). Treatment increased the
genotypes studied, in contrast to the findings of Saini et al. (1983),
oxidation of probes incorporated into the membrane in both
who reported that the embryo sacs were completely absent
genotypes, but to a different extent. On average, the proportion
or formed abnormally with incomplete cellular organization
of oxidized C11-BODIPYTM rose by 18% and 48% in Plainsman
and altered ovary development (reduced nucellus development,
V and Cappelle Desprez, respectively (Figure 8H).
overproliferated integuments) in pistils subjected to continuous
exposure to 30◦ C for 3 days during meiosis. The effect of water
Correlation Between Reactive withdrawal on wheat ovule development is rather controversial.
Compounds and Fertility Loss As found here, Saini and Aspinall (1982) reported that water
Very strong or strong negative correlations were found withdrawal per se had no effect on ovule development. In
between the fertility ratio of the genotypes and the total ROS contrast, Onyemaobi et al. (2017) considered that female
content (r = −0.85), extracellular H2 O2 content (r = −0.94), reproductive organs could be one of the major contributors to
intracellular H2 O2 content (r = −0.84), OH• and ONOO− low seed set in wheat stressed during meiosis. It is important
content (r = −0.91), mitochondrial O2 •− content (r = −0.91), to note that the male and female gametophytic processes in
cytoplasmic O2 •− content (r = −0.47) and lipid peroxidation Triticum aestivum are not synchronized after meiosis and that
(r = −0.60). A very strong positive correlation was found between the differentiation of the octonucleate embryo sac (female
the cytoplasmic O2 •− content and lipid peroxidation, while very gametophyte) proceeds far more rapidly than that of its male
strong correlations were detected between the generation of counterpart (Tímár et al., 1997). It can be assumed that the
intracellular H2 O2 (r = 0.98), mitochondrial O2 •− (r = 0.93), 7-celled female gametophyte was already formed by the time
OH• ONOO− (r = 0.92) and extracellular H2 O2 . A moderate the microspores entered the binucleate stage of development,

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

FIGURE 8 | Localization of (A,C,E,G) ROS, RNS and peroxidized lipids and (B,D,F,H) relative fluorescence observed after Ampliflu Red (A,B), DHR 123 (C,D), DAF
FM-DA (E,F) and C11-BODIPYTM 581/591 (G,H) labeling in stigma papilla cells of control and HD-stressed Plainsman V and Cappelle Desprez wheat plants at
anthesis. Ampliflu Red, DHR 123 and DAF FM-DA indicate the generation of extracellular H2 O2 , intracellular H2 O2, and nitric oxide, respectively. The intensity of
C11-BODIPYTM 581/591 labeling reveals the extent of lipid peroxidation. Arrows, specific C11-BODIPYTM signal from the plasma membrane; arrowheads,
non-specific autofluorescence from the cell wall. In each histogram, letters above columns indicate significant differences between means at the P ≤ 0.05 level of
probability. Base, lower half of the spike; top, upper half of the spike. Bar represents (A,C,E,G) 10 µm and (G inset) 1 µm.

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

which means that the development of the female gametophyte was demonstrated here, that structural anomalies triggered by
was accomplished by the second day of HD stress, which is why the HD-induced generation of ROS and RNS may stand in
the treatment had no negative effect on the structure of the ovules. the behind of reduced stigma function and female-dependent
No reduction in anther length was observed after HD fertility loss. In photosynthetic tissues, the main sources of
stress. Nevertheless, in agreement with reports of the pollen ROS are the chloroplast and the peroxisome, especially when
viability-reducing effect of high temperature or drought (De conditions are unfavorable (Waszczak et al., 2018). However,
Storme and Geelen, 2014), HD stress severely reduced pollen as photosynthesis does not occur in stigmatic papilla cells,
viability by 63% and 81%, in the basal and top halves, respectively, major ROS-generating processes such as the reduction of O2
of treated spikes of the sensitive genotype. The competition for in photosystem I or photorespiration in the peroxisomes are
assimilates between the upper and basal spikelets is a well-known absent. The potential ROS-generating regions in papilla cells are
phenomenon that could be more exacerbated when the plants therefore the mitochondria, the glyoxysomes and the plasma
encounter drought stress (Chen et al., 2013), high temperature membrane-cell wall-apoplast system (Noctor et al., 2014; Singh
stress (Fu et al., 2016) and their combination. et al., 2016; Waszczak et al., 2018). Fluorescent ROS indicators
The analysis of yield components revealed that Cappelle provide powerful tools for the investigation of oxidative stress
Desprez suffered significantly greater loss in plant production in living cells. Nevertheless, the majority of these probes have
after treatment, so this variety was considered as sensitive to HD limitations due to their insufficient ROS specificity, which should
co-stress. The data indicate that the stress-induced damage to be taken into account when interpreting the results (Ortega-
stigmatic papilla cells in Cappelle Desprez, which was verified Villasante et al., 2016). The present study confirmed that the
by the results of anatomical observations in the present study, stigmatic papillae of monocotyledonous wheat generate high
strongly contributed to the decrease in function and fertility. amounts of ROS (O2 •− , OH− , H2 O2 ) and RNS (ONOO− , NO)
Spike fertility, which determines grain number and sink strength, at anthesis. These results are in accordance with the findings of
is a crucial factor in wheat yield potential (Reynolds et al., 2009). McInnis et al. (2006), Serrano et al. (2010), and Zafra et al. (2016)
Heat and drought stress significantly decrease fertility (Prasad who observed the accumulation of ROS/H2 O2 in dicotyledonous
et al., 2011), which is generally considered to be the consequence angiosperms. The assessment of general cellular oxidative stress
of the damage sustained by the male gametophyte, while the role using H2 DCFDA indicated a good correlation with fertility loss,
of pistil dysfunction in yield loss is somewhat underestimated. implying that the high level of oxidants detected in the stigmatic
The loss observed in the fertility of the HD-stressed sensitive papillae was located in the top half of Cappelle Desprez spikes.
genotype in this study highlighted the fact that not only the The amount of oxidants in Plainsman V rose to a smaller extent,
extensively studied meiotic processes, but also development which did not lead to fertility loss.
of sexual organs taking place during gametogenesis is highly The ROS metabolism in plant cells is an intricate network
sensitive to a changing climate. Apart from the great sensitivity involving many enzymatic and metabolite elements (Noctor
shown by pollen development to heat and drought stress (Saini et al., 2015). The first type of ROS generated by the electron
and Aspinall, 1981; Saini et al., 1984; Lalonde et al., 1997; Jäger transport chain in mitochondria is superoxide (Rhoads et al.,
et al., 2008; for reviews, see Dolferus et al., 2011; De Storme 2006). Unfortunately, the monitoring of superoxide with
and Geelen, 2014), the results of the pollination experiment DHE and MitoSOX Red is impaired by the two-electron
demonstrated that the damage to female reproductive organs oxidation of these dyes by other oxidants (Wang et al., 2013),
induced by HD stress reduced their functionality and was making these probes unsuitable for the selective detection of
responsible for 34% of gross fertility loss. It might be expected O2 •− . Nevertheless, the similar sensitivity and the organelle
that decreased fertility would be negatively correlated with TSW selectivity of these probes make them ideal for the quantitative
due to compensation. This was not true of Plainsman V, in which comparison of the oxidants present in the mitochondria
neither fertility nor TGW was influenced by the treatment. In and cytoplasm (Zielonka and Kalyanaraman, 2010). The
contrast, the fertility and TGW of Cappelle Desprez dropped control level of these oxidants was fourfold higher in the
significantly both in the basal and top floret positions as a mitochondria compared to the cytoplasm, confirming the
consequence of HD which indicates that this variety was unable leading role of mitochondria in ROS generation. HD stress
to compensate for the reduced grain number by an increase triggered a sharp rise in mitochondrial oxidant generation
in grain weight. Moreover, although the duration of grain fill exclusively in Cappelle Desprez, while the cytoplasmic amount
was shortened by 10 and 14 days in the tolerant and sensitive of these compounds rose significantly in both genotypes,
genotype, respectively, the former gave a plant production similar irrespective of floret position. This suggests that although
to the control, while the production of the latter dropped in both Plainsman V possesses a more efficient mitochondrial electron
spike halves. Prasad et al. (2011) reported a similar decrease in transport chain and/or ROS scavenging system than Cappelle
the number of days to physiological maturity of bread wheat Desprez, there are other cytoplasmic oxidant-generating
cultivars under a combination of drought and high temperature mechanisms which produce similar levels of oxidizing agents
stress applied at heading. in the cytoplasm of both varieties. A very strong positive
As oxidative stress is an important source of damage when correlation was found between the cytoplasmic O2 •− content
plants face high temperature and water shortage simultaneously and lipid peroxidation.
(Zandalinas et al., 2018), it can be assumed that ROS-mediated Superoxide is converted by superoxide dismutase enzyme
injury is a major factor contributing to structural changes. It (SOD) into hydrogen peroxide, a ROS with a long lifespan

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

(Waszczak et al., 2018). H2 O2 is able to diffuse through peroxide, a precursor molecule of OH• , closely mirrored the
membranes either alone (Lim et al., 2016) or facilitated by presence of the APF signal in the present experiment, which
aquaporins (Bienert and Chaumont, 2014), and may thus reach was not true for the precursors of peroxynitrite, NO and
other parts of the cells. In the present study DHR 123 was used O2 •− . These data imply that the APF signal is potentially
to monitor intracellular hydrogen peroxide, but as it also reacts more indicative of hydroxyl radicals than peroxynitrite in
with peroxynitrite (Crow, 1997), the results must be handled this experiment.
with care. DHR 123 diffuses freely into the cells, where it is The nitric oxide content and fertility of Cappelle Desprez
oxidized to Rhodamine 123, which selectively stains mitochondria were found to be lower in the top than in the basal halves
(Johnson et al., 1980). Hence, although the fluorescent signal of control spikes. After HD treatment, both values dropped in
is detected in the mitochondria, it represents the H2 O2 and this genotype, irrespective of floret position, while Plainsman
peroxynitrite concentration of the whole cell. The results showed V showed no significant change. An increasing number of
that the cytoplasmic concentration of these compounds was only scientific papers report the significance of NO in fertilization.
elevated in the top half of Cappelle Desprez spikes. In non- Seligman et al. (2008) demonstrated the NO-generating activity
photosynthesizing cells, the hydrogen peroxide level may be of stigmatic tissue in Arabidopsis. Moreover, an Arabidopsis
elevated not only by mitochondrial activity, but also through mutant proven to be defective in NO production showed reduced
the glyoxylate cycle in glyoxysomes. This pathway allows the fertility, which was restorable using a NO donor compound
turnover of membrane lipids as well as the synthesis of various (Guo et al., 2003). The present results confirm the proposed
hormones playing important roles in the stress response, like link between the HD stress-triggered drop in nitric oxide
indole acetic acid, jasmonic acid and salicylic acid (Corpas et al., content and reduced fertility in monocotyledonous plants. This
2015). Higher expression of acyl-CoA oxidase, an enzyme that link may be the role that nitric oxide plays in pollen tube
generates H2 O2 in the glyoxylate cycle, was reported in drought- guidance. Prado et al. (2008) showed that NO acts as a negative
treated Arabidopsis plants (Bray, 2002). It can be hypothesized chemotropic agent of pollen tube growth, providing a tool
that the glyoxylate cycle became more active in the upper for maternal tissues to route pollen tubes toward the ovule.
florets of Cappelle Desprez, contributing to the elevated H2 O2 Positive correlation between fertility and NO content of stigmatic
level in papilla cells. Increased amounts of apoplastic H2 O2 , papilla cells indicates that this compound promotes successful
determined with Ampliflu Red, were observed exclusively in fertilization in wheat.
treated Cappelle Desprez papilla cells. Apoplastic ROS is generated Oxidative damage during abiotic stress originates mainly from
actively by enzymatic mechanisms, such as apoplastic polyamine the alteration of lipids and proteins by oxidative compounds
oxidases and respiratory burst oxidase homologs localized in in the cell (Anjum et al., 2015). The ratio of oxidized to
the plasma membrane (reviewed by Waszczak et al., 2018). reduced Cll-BODIPY 581/591, a fluorescent membrane lipid
According to the literature, apoplastic hydrogen peroxide is homolog, increased to a higher extent in Cappelle Desprez
involved in drought and salt stress acclimation (Miller et al., 2010), than in Plainsman V after HD treatment. Moreover, a very
although a more important role is proposed in intercellular signal strong positive correlation was found between the cytoplasmic
transduction (Choudhury et al., 2017). The higher apoplastic O2 •− content and lipid peroxidation. It should be noted that
levels of H2 O2 in Cappelle Desprez may be explained by the the oxidation of Cll-BODIPY 581/591 can only be initiated by
occurrence of more severe water shortage in this genotype, a variety of oxy-, peroxy-, or hydroxyl radicals, but not by
which may induce elevated amounts of signaling molecules. superoxide, nitric oxide, or hydroperoxides (Pap et al., 2000),
Very strong correlations were found between the generation so the fluorescent signal intensity of the probe is not adequately
of intracellular H2 O2 , mitochondrial O2 •− , OH• , ONOO− , and proportional to lipid peroxidation. However, this fluorophore can
apoplastic H2 O2 . be used to estimate the general oxidation of membrane lipids.
The amounts of highly reactive oxidative and nitrosative It can be assumed that the hostile microenvironment
compounds, hydroxyl radical (OH• ) and peroxynitrite induced by the generation of ROS and ONOO− and the
(ONOO− ), respectively, were estimated using APF (Setsukinai decrease in NO production prevented successful pollen–pistil
et al., 2003). The results showed that the amount of these interactions, resulting in fertilization failure in a significant
compounds elevated only in Cappelle Desprez papillae number of Cappelle Desprez florets. Although there seems to
following HD stress, especially in those located in the top be a close correlation between high levels of fluorescence from
half of the spikes. In living cells, hydroxyl radical (OH• ) is ROS-sensitive probes and fertility loss, the exact cause and
formed from H2 O2 through the Fenton reaction catalyzed mechanisms of ROS-derived injury and the drop in nitric oxide
by iron and other transition metals (Rhoads et al., 2006). content are still not clear due to the low specificity of the
Despite its short lifetime, OH• has a significant role in lipid fluorescent probes currently available.
peroxidation (Pham-Huy et al., 2008; Noctor et al., 2015).
Peroxynitrite (ONOO− ), another highly reactive radical which
can oxidize APF, is generated by the reaction of O2 •− with CONCLUSION
NO (Arasimowicz-Jelonek and Floryszak-Wieczorek, 2011).
This short-lived RNS takes part in the oxidation and nitration This is the first report on the effect of HD co-stress on female
of various molecules, such as DNA, lipids, and proteins reproductive cells and organs in wheat. Overall, this research
(Vandelle and Delledonne, 2011). The amount of hydrogen revealed that HD co-stress reduced the RWC of wheat plants

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Fábián et al. Heat-and-Drought Reduced Stigma Functionality in Wheat

and increased ROS and ONOO− generation, decreased NO out statistical analysis and wrote the manuscript. All authors
production and enhanced lipid peroxidation in stigmatic papilla read and reviewed drafts of the manuscript and approved the
cells. These changes induced alterations in the morphology final manuscript.
and anatomy of female reproductive organs and shortened the
duration of gametogenesis and grain filling, with the combined
effect of significantly reduced fertility and plant production to FUNDING
an extent dependent on floret position and tolerance. Further
investigations will be needed to shed light on the genetic The work was financed by a grant from the National Research,
background of the differences observed between the studied Development and Innovation Office – NKFIH, (NKTH-OTKA
genotypes. A better understanding of the effect of HD stress and K108644) and by grants from the Hungarian Academy of
the mechanisms of tolerance may lead to the development of Sciences (GENPROF IF-18/2012, KEP-5/2016-2018).
wheat genotypes suitable for future climatic conditions.

ACKNOWLEDGMENTS
DATA AVAILABILITY
The authors thank Erika Gondos, Barbara Krárné Péntek, and
All datasets generated for this study are included in the Szilvia Fodor for their excellent technical assistance.
manuscript and/or the Supplementary Files.

SUPPLEMENTARY MATERIAL
AUTHOR CONTRIBUTIONS
The Supplementary Material for this article can be found online
KJ conceived and designed the experiments. AF, ES, GS-E, at: [Link]
BB, and KJ, performed the experiments. AF and KJ carried full#supplementary-material

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