influence by iso-osmotic stress of PEG and NaCl and their relative mitigation effect of Se
Lan CY, Lin KH, Chen CL, Huang WD, Chen CC (2020). Comparisons of chlorophyll fluorescence and physiological characteristics of wheat seedlings influenced by iso-osmotic stresses from polyethylene glycol and sodium chloride. Agronomy, 10(3): 325
Abstract
This study aimed to distinguish the effects of the osmotic and ionic effect caused by
salt stress and to evaluate the contribution of 22 μM Se treatment on both effects on Wheat
(Triticum aestivum L.) cultivar Taichung SEL.2. We hypothesized that TCS2 is more vulnerable to the osmotic stress according to its’ salt-tolerance property and that 22 μM
Se exert a beneficial effect to both stresses. In order to test the hypothesis, the osmotic
agents of polyethylene glycol (PEG) and NaCl were used in three iso-osmotic
concentrations with final osmotic potentials (OP) of -1.05 MPa (24% (w/v) PEG and 200
mM NaCl), -1.33 MPa (26.5% (w/v) PEG and 250 mM NaCl) and -1.57 MPa (29% (w/v)
PEG and 300 mM NaCl). The more-stabilized chlorophyll fluorescence (ChlF) parameters (maximal quantum yield of phothsystem II (Fv/Fm), effective quantum yield
of photosystem II(ΦPSII), non-photochemical quenching of PSII (NPQ), and the
less-extensive degradation of photosynthetic pigments (total chlorophyll and carotenoids)
were observed under NaCl-treated seedlings. Ascorbate peroxidase (APX) activity was
performed better and the less accumulation of malondialdehyde (MDA) were identified
on NaCl-treated seedlings. The elongation of shoots of NaCl-treated seedling was also
preserved on NaCl-treated seedlings. The results above supported the hypothesis that
TCS2 is more vulnerable to osmotic stress than the toxic effect of ion under 3 levels of
iso-osmotic concentrations created by PEG and NaCl. Nevertheless, contrary to our
hypothesis, a disadvantageous effect of 22 μM Se had been observed between Se
treatments (No added Se and 22 μM Se) in the ChlF performances of Fv/Fm, ΦPSII and
NPQ and APX activity, photosynthetic pigments, resulting in a higher accumulation of MDA concentration in seedlings treated with 22μM Se. The effective treatment
concentration of Se required furthering experimentation.
Keywords: wheat; salt stress; osmotic stress; selenium; chlorophyll fluorescence; APX activity
Introduction
Salinity has impacted wheat (Triticum aestivum L.) production around the world with increasing frequency and intensity. It was estimated that more than 69% of the total wheat
production had been seriously influenced by high salinity (Isayenkov, 2012). Therefore,
it is crucial to understand the mechanisms by which wheat perceive and adapt to salt stress
so that the situation can be adequately improved.
Plant response to salinity at different time scales called a two-phase growth response
(Munns and Sharp, 1993). The first phase was water stress phase or osmotic phase, which
happened within minutes to hours, caused by the salt outside the roots inhibited the water
absorption of plant, resulting in “physiological drought” (Filek et al., 2012). The second
phase was the toxic effects of ions, which takes days to progress (Munns, 2002). As a
result, salt stress could trigger many common reactions in a plant similar to that of drought
stress. Therefore, it is difficult to distinguish between the effect of drought and salt stress
and their relative importance to plant. Indeed, what is the primary mechanism which
governed a crop’s ability to cope with salt stress? Is it the capacity of crop to confront
drought, or the ability to against toxic effects of ions in salt stress?
There are many studies aimed to answer this question. In those studies, the
iso-osmotic potential of NaCl has been most commonly generated by polyethylene glycol
(PEG), a widely used osmotica to induce water stress in order to simulate low water
potential due to dry soil (Almansouri et al., 2001), as it is a non-ionic, neutral and
water-soluble polymer which does not penetrate the roots (Sayar et al., 2010). The results of
those studies vary depending on tested species/varieties and the development stages. In
the results of some studies, PEG was more harmful than NaCl treatments. It occurred in
the cases of germination stage of durum wheat (Almansouri et al., 2001) and Hordeum
species (Huang and Redmann, 1995), and on seedling stage in wheat (Filek et al., 2012;
Sayar et al., 2010). However, Muranaka et al. (2002a, b) have revealed that NaCl
treatments were more harmful than that of PEG treatments on the seedling stage of wheat.
Some studies compared between sensitive and resistant varieties and reported that the
salt-resistant varieties are more vulnerable to water deficits while ‘ion excess’ might
prevailing over in the sensitive types (Greenway and Munns, 1980; Sharma et al., 1984).
The salinity resistance also varies with the development stage of the plant (Borsani et al.,
2001; Lutts et al., 1995). In the study of Sayar et al. (2010), the seedling stage was more
susceptible to NaCl than to PEG under iso-osmotic potential treatments than germination.
It is obvious that there are species/genotypes/stages-specific responses to salinity
(Shannon, 1997). Crops/varieties/developmental stages suffered more in NaCl treatment
when they are unable to prevent salt entry (gene-related) (Chaves et al., 2009; Munns,
from reaching the toxic levels in leaves (Munns, 2002). However, if
crops/genotypes/developmental stages could tolerance the resulting ion concentrations
caused by NaCl treatment (Yeo, 1983), Na+ and Cl- may further serve as an osmoticum
(Hsiao et al., 1976) to produce a rapid osmotic adjustment (Prat and Fathi‐Ettai, 1990) so
that the shoot turgor could be maintained. The osmoregulation created by ionic NaCl was
much faster as well as less energy and carbon-demanding than organic solutes involved
in osmotic adjustment created by PEG (Pérez‐Alfocea et al., 1993), which is the main
reason why PEG treatments were more harmful than those of NaCl treatments in some
varieties.
To evaluate the relative influence of osmotic and ionic stress caused by salt stress, the
biochemical, physiological and morphological responses can be applied to get insight to
the mechanisms involved (Slama et al., 2008). Basically, the enzymatic and
non-enzymatic defense system and the photosynthetic system will be inhibited, the lipid
peroxidation products will be increased, and the growth will be reduced (Zörb et al., 2019).
The biochemical analysis provides information regarding the process above. Nevertheless,
it is time-consuming and requires complicated technical support. However, the
chlorophyll fluorescence (ChlF) of PSII can provide effective physiological changes as a
simple and rapid procedure (Sayar et al., 2008; Siringam et al., 2009). In this study, the
measurements of ChlF were conducted for early identification, following by growth
analysis and the measurements of biochemical (APX activity, lipid peroxidation product:
malondialdehyde (MDA) and photosynthetic pigments) analysis.
Selenium (Se) is considered a beneficial element to plants (Pilon-Smits et al., 2009).
Previous studies indicated that Se can delay senescence (Djanaguiraman et al., 2005; Xue
et. al.,2001) and promote the vegetative and reproductive growth of plants (Hajiboland et
al., 2012). Studies reported that Se mitigated disadvantageous phenomena caused by
various stressful situations, such as drought (Xiaoqin et al., 2009; Nawaz et al., 2015),
and salt stress (Mona et al., 2017; Diao et al., 2014). Therefore, a positive effect of Se
related to the effects of osmotic and ionic effect caused by salt stress was expected.
Wheat (Triticum aestivum L.) cultivar Taichung SEL. 2 (TCS2), one of the most
widely cultivated cultivars in Taiwan, appeared to be a salt-tolerant cultivar (Lan et al.,
2019). However, the mechanisms that governed its ability to cope with such stress
remained unclear. Hence, the purpose of this study was to understand the biochemical and
physiological responses of TCS2 under iso-osmotic potentials created by PEG and NaCl
in order to distinguish its abilities and mechanisms to cope with salt stress. We
hypothesize that TCS2 is more vulnerable to osmotic stress than the toxic effect of ion
under 3 levels of iso-osmotic concentrations created by PEG and NaCl and that 22 μM
Se exert a beneficial effect to both stresses.
Materials and methods
Plant and growth conditions
Wheat (Triticum aestivum L.) cultivar Taichung SEL. 2 (TCS2), one of the most
widely cultivated wheat cultivars in Taiwan, was used in this study. Seeds used in the
present study were obtained from the Department of Agronomy, National Taiwan
University (Taipei, Taiwan). The seeds were sterilized with 1% hydrogen peroxide for 5
min, washed with distilled water, and germinated in Petri dishes on wetted filter paper at
25 °C in the dark. After 24 h of incubation, uniformly germinated seeds were selected
and cultivated in 150 mL beakers containing complete Hoagland’s nutrient solution
(Hoagland and Arnon, 1950), which was replaced every 3 days. Hydroponically
cultivated wheat seedlings were raised in growth chambers with fluorescent lamp lighting
at 25 and 20 °C during the day and night, respectively, under a 12 h photoperiod. The photosynthetic photon flux density (PPFD) was uniformly set to 300 μmol m-2 s-1.
Experimental treatments
Se was added in the form of Sodium selenite (Na2SeO3, Sigma-Aldrich Chemie
GmbH, Taufkirchen, BY, Germany) to the nutrient solution (pH = 4.6) with the treated
concentration of 22 μM once the germinated seeds were cultivated in 150 mL beakers.
No treatment (Control) consisted of the nutrient solution without Se supplementation.
Hydroponically grown seedlings that had reached stage Z1.0 (Zadoks et al., 1974) on day
6 were treated with the osmotic agents of polyethylene glycol (PEG-6000, Merck) and
NaCl (Sigma). These osmotic agents were used in three iso-osmotic concentrations based
on the treatments of Almansouri et al. (2001) with final osmotic potentials of -1.05 MPa
(24% (w/v) PEG and 200 mM NaCl), -1.33 MPa (26.5% (w/v) PEG and 250 mM NaCl)
and -1.57 MPa (29% (w/v) PEG and 300 mM NaCl). The experiment was independently
performed three times for a randomized design of growth conditions.
Growth analysis
Shoot height and root length were measured with a ruler before the measurements of
chlorophyll fluorescence (ChlF) and sample collection.
Measurements of ChlF
Fluorescence parameters in seedling leaves were determined after PEG and salt
treatments. ChlF was measured in the middle portion of the first leaf of each seedling
taken at ambient temperature with Chl fluorometer imaging-PAM (Walz, Effeltrich,
Germany). Actinic light and saturating light intensities were set to 185 and 7200 μmol m
-2s-1 of photosynthetically active radiation (PAR), respectively. The minimal (F0) and
maximal (Fm) ChlF, the maximum quantum yield of PSII (Fv/Fm), the effective quantum yield of PSII (ΦPSII) and the non-photochemical quenching of PSII (NPQ) were measured
and calculated according to previously described methods (Kramer et al., 2004; Van and
Snel, 1990).
Measurement of APX activities
APX activities were measured base on the method of Nakano and Asada (1981).
Briefly, 0.1 g of the latest newly expanded leaf was placed in 2 mL of sodium phosphate
buffer (50 mM, pH 6.8) in an ice bath for extraction and centrifuged at 4°C and 12,000
rpm for 20 min. The supernatant (0.1 mL) was collected, followed by the sequential
addition of 2.7 mL of potassium phosphate buffer (150 mM, pH 7.0), 0.4 mL of
ethylenediaminetetraacetic acid (EDTA, 0.75 mM), 0.5 mL of H2O2 (6 mM), 0.5 mL of
H2O, and 0.5 mL of ascorbate (1.5 mM) and then mixed well. The absorbance at 290 nm
of the sample solution was determined every 15 s for 1 min using a spectrophotometer
(Hitachi U3010, Tokyo, Japan). The blank containing the same mixture with no enzyme
extract was also measured.
Determination of the photosynthetic pigment concentrations
The photosynthetic pigment concentrations were determined using the method of
Yang et al. (1998). Briefly, 0.01 g of lyophilized sample powder was extracted with 12
mL of an 80% acetone solution, and then centrifuged at 4500 rpm for 5 min. The
supernatant of the sample extract was tested to determine the absorbance extents of Chl
a, Chl b, and carotenoids (Car) in acetone at 663.6, 646.6, and 440.5 nm, respectively.
Determination of the MDA concentration
MDA was determined using a previously described method of Heath and Packer
(1968). Briefly, lyophilized sample powder (0.03 g) was mixed with 1 mL of 5% TCA,
and then centrifuged at 10,000 rpm and 20 °C for 5 min. The supernatant (250 μL) was
added to 1 mL of 0.5% thiobarbituric acid (TBA) which was made up with 20% TCA.
The mixture was placed in a water bath at 95°C for 30 min, and then immediately cooled
in an ice bath. The reaction mixture was centrifuged at 3000 rpm and 20°C for 10 min,
and the absorbance was determined at 532 and 600 nm. The blank was the same reaction
mixture with no sample extract.
Statistical analyses
All measurements were evaluated for significance using an analysis of variance
(ANOVA) followed by a least significant difference (LSD) test and t - test at p < 0.05.
All statistical analyses were conducted using R i386 3.5.1 software
(https://cran.r-project.org/bin/windows).
Results
Growth analysis
There was no significant difference between Se treatments (No added Se and 22 μM
Se) in all levels of three corresponding iso-osmotic potentials creating by PEG & NaCl
in the performance of shoot height (Fig. 4-1A). Therefore, the results without Se treatment
were further discussed in the following content (Fig. 4-1B).
Shoot heights of seedling with PEG treatment declined dramatically from 24.3 to 12.6
cm with treatment concentration of 24% (w/v) PEG and then decreased gradually to 11.6
cm with the final treatment concentration of 29% (w/v) PEG. A similar declining trend
was observed in NaCl-treated seedlings. However, NaCl-treated seedlings were always
significantly longer (p < 0.05) than those of PEG-treated seedlings under iso-osmotic
potential treatments.
ChlF
A disadvantageous effect of 22 μM Se had been observed between Se treatments (No added Se and 22 μM Se) in the ChlF performances of Fv/Fm (Fig. 4-2A), ΦPSII and
NPQ (Fig. 4-3A) , which was contrary to our hypothesis. Therefore, the results without
Se treatment were further discussed in the following content (Fig. 4-2B; Fig. 4-3B).
Fv/Fm andΦPSII in leaves, determined after dark adaption and under illumination, are
the indexes of the maximum and effective quantum yield of PSII, respectively. They are
widely used to estimate the status of plant under stress (Sun et al., 2006). Fv/Fm in leaves
of seedlings with PEG-treatment declined dramatically with the increasing PEG
concentrations from 0.781 to 0.019 (p < 0.05). However, Fv/Fm in leaves of seedlings with
NaCl-treatment maintained stable until the NaCl concentration exceeded 250 mM. As a
result, Fv/Fm valves in leaves of NaCl-treated seedlings were always significantly higher
(p < 0.05) than those values in leaves of PEG-treated seedlings under iso-osmotic
potential treatments (p < 0.05) (Fig. 4-2B). ΦPSII in leaves of seedlings with
PEG-treatment declined with the increasing PEG concentrations from 0.526 to 0.013 (p < 0.05).
ΦPSII in leaves of seedlings with NaCl-treatment showed the same trend with those of
PEG-treated seedlings (p < 0.05), with NaCl-treated seedlings significant higher than that
of PEG-treated seedlings under the highest osmotic potentials of -1.57 Mpa (p < 0.05).
NPQ represents the non-photochemical quenching of PSII. NPQ in leaves of seedlings with PEG-treatment declined dramatically with the increasing PEG concentrations from
0.22 to 0.00 (p < 0.05), while NPQ in leaves of seedlings with NaCl-treatment increased
dramatically in 200 mM than gradually decreased to the similar value of CT. NPQ valves
in leaves of NaCl-treated seedlings were always significantly higher (p < 0.05) than those
values in leaves of PEG-treated seedlings under iso-osmotic potential treatments (p < 0.05)
(Fig. 4-3B).
APX activities
A disadvantageous effect of 22 μM Se had been observed between Se treatments (No added Se and 22 μM Se) in APX activity (Fig. 4-4A), which was contrary to our
hypothesis. Therefore, the results without Se treatment were further discussed in the
following content (Fig. 4-4B).
The APX activities of PEG and NaCl-treated seedling both showed descending trends
with significant higher activities in NaCl-treated seedling than those of PEG-treated
seedlings under the same iso-osmotic potential (p < 0.05) (Fig. 4-4B).
Photosynthetic pigments
A disadvantageous effect of 22 μM Se had been observed between Se treatments (No added Se and 22 μM Se) under three corresponding iso-osmotic potential creating
by PEG & NaCl in photosynthetic pigments (Fig. 4-5A) , which was contrary to our
hypothesis. Therefore, the results without Se treatment were further discussed in the
following content (Fig. 4-5B).
The Chl a, b, a+b concentrations of PEG and NaCl-treated seedling all showed
descending trends with significant higher concentraions in NaCl-treated seedling than
those of PEG-treated seedlings under the same iso-osmotic potential (p < 0.05) (Fig.
4-5B). The concentration of Car decreased significantly once PEG and NaCl were applied
to seedlings, but there was no significant difference with an increase in the PEG and NaCl
significantly higher (p < 0.05) than those of PEG-treated seedlings under iso-osmotic
potential treatments (Fig. 4-5B).
MDA concentration
Seedlings with 22 μM Se treatment accumulated more MDA concentration than the
seedlings without Se treatment under three corresponding iso-osmotic potential creating
by PEG & NaCl (Fig. 4-6A), which was contrary to our hypothesis. Therefore, the results
without Se treatment were further discussed in the following content (Fig. 4-6B).
MDA concentration of seedlings with PEG treatments increased dramatically from
13.5 nmol g-1 DW (CT) to 116.7 nmol g-1 DW (29% (w/v) PEG) (p < 0.05), while the
MDA concentration in NaCl-treated seedlings were just increase slightly from 13.5 nmol
g-1 DW (CT) to 33.2 nmol g-1 DW (300 mM NaCl) with no significant difference between
NaCl treatments. The MDA concentration of PEG-treated seedlings were always
significantly higher (p < 0.05) than those of NaCl-treated seedlings under iso-osmotic
potential treatments (Fig. 4-6B).
Discussion
A disadvantageous effect of 22 μM Se had been observed between Se treatments (No added Se and 22 μM Se) in the ChlF performances of Fv/Fm (Fig. 4-2A), ΦPSII & NPQ
(Fig. 4-3A), APX activity (Figure 4-4A) and photosynthetic pigments (Figure 4-5A), resulted in a higher accumulation of MDA concentration in seedlings treated with 22μM
Se (Figure 4-6A).
Although a beneficial micronutrient, Se exerts a dual effect in plants (Djanaguiraman
et al., 2005): It can stimulate plant growth and provide beneficial effects at low
concentration, but it is harmful to plants at higher concentrations. Positive effects of Se
depend on its form, dose and the chosen plant genotype (Sieprawska et al., 2015).
According to the results of our preliminary study for wheat (T. aestivum L.) cultivar
Taichung SEL. 2, the growth rate of wheat seedlings was strongly retarded at 10 mg Se
L−1 (294 μM), but was not promoted at 0.5 mg Se L−1 (14.7 μM). Se at 1 mg L−1 (29 μM)
might exceed the plant’s threshold, resulting in a slightly disadvantageous effect on the
plant. Therefore, 0.75 mg Se L−1 (22 μM) was an appropriate concentration for Se
treatment in this study. In Se & Salt exp. (chapter 3), the protective effect of 22 μM Se on
the growth and physiological traits of wheat seedlings under salt stress had been proved.
However, the toxic effect of 22 μM Se was observed in Se & Salt/PEG exp. (chapter 4).
It is obvious that, apart from its form and dose, some other environmental factors may
also contribute to the effects of Se. In fact, the experiments of Se & Salt exp. (chapter 3)
and Se & Salt/PEG exp. (chapter 4) were conducted in different growth chamber. The
seedlings of Se & Salt exp. (chapter 3) were incubated in the growth chamber 1 while the
seedlings of Se & Salt exp. (chapter 4) were grown in the growth chamber 2. The
conflicting results may be due to differences in the light source or microenvironment
conditions between growth chambers 1 and 2. Therefore, a further experiment was needed to confirm the effective treatment concentration of Se.
Contrary to our hypothesis, a disadvantageous effect of 22 μM Se has been observed
between Se treatments (No added Se and 22 μM Se). Therefore, the following discussion
was focusing on the results of “No added Se” treatments.
In order to identify the relative influences between water deficits and ion toxic effects
resulting from salt stress, comparisons between three levels of iso-osmotic concentrations
of PEG and NaCl treatments have been done. The treatment concentrations of PEG and
NaCl were chosen according to visual symptoms of osmotic and ion-toxic effect on wheat
leaves. The noticeable sigh of the water deficits are leaf wilting and rolling due to stomatal
closure created by PEG (Filek et al., 2012), while the ion excess symptom resulting from
NaCl treatments are leaf yellowing or death of older leaves (Munns, 2002). The treatment
period was set to allow the water relations and toxic effects of ions symptoms becoming
apparent visible while causing no irreversible damage to wheat (Filek et al., 2012). The
ChlF of PSII was than measured as an early identification.
The Fv/Fm, NPQ, Y(NPQ) and Y(NO) values were statistically more influenced by
osmotic stress created by PEG than by NaCl treatment in all levels of iso-osmotic
concentrations, while ΦPSII values also showed the same trend in the highest iso-osmotic
concentration of -1.57MPa. (Fig. 1B, 2B) The result of ChlF measurements indicating
that the PSII efficiency were significantly affect and damage by the induced water stress.
Drought and salt stress affect photosynthesis either by diffusion limitations through the
stomata (stomatal closure) or by the influence of photosynthetic metabolism (Chaves et
al., 2009; Muranaka et al., 2002a). It may be highly related with the osmoregulation
process under drought and salt stress (Prat and Fathi‐Ettai, 1990). Under osmotic stress,
the osmotic adjustment was accomplished by synthesizing organic compatible solutes
such as organic acids, amino acids, sugars and polyols (Hellebusi, 1976; Sayar et al.,
2010), which take time to build up (Hsiao et al., 1976). Whereas under salt stress, turgor
could be maintained by uptake of ions (Na+ and Cl-) (Sharma et al., 1984), and the cost
is much lower than the ATP needed to synthesize organic solute (Munns, 2002). It might
also be the results that the treatment time was not long enough to allow the organic solutes
indifference values ofΦPSII between isoosmotic concentrations of PEG and NaCl in -0.58 and -1.33Mpa indicated that the capability of PSII under the absorbed irradiance of
185 μmol m-2s-1 of PAR in the photochemical reaction were similar. The result was
supported by Muranaka et al. (2002a) and Flagella et al. (1998) who had discovered that
supported by Muranaka et al. (2002a) and Flagella et al. (1998) who had discovered that