Exogenous S-Methylmethionine Alleviates Salinity Stress by Modulation
of Physiological Processes in Canola (Brassica napus)
Laszlo Fodorpataki1,2*, Katalin Molnar3, Bernat
Tompa1 and Csaba Bartha3
1Hungarian Department of Biology and Ecology,
Babes-Bolyai University, RO-400084 Cluj-Napoca, Romania
2Laboratory of Plant and Microbe Biotechnologies and
Environmental Interactions, Centre of Systemic Biology, Biodiversity and Bioresources,
Babes-Bolyai University, RO-400084 Cluj-Napoca, Romania
3Department of Horticulture, Sapientia Hungarian
University of Transylvania, RO-540485 Targu Mures, Romania
*For correspondence: lfodorp@gmail.com
Received 07 May 2020; Accepted 27 August 2020; Published 10 December
2020
Abstract
Canola is a
moderately salt tolerant plant, high salinity inhibits germination of seeds,
vegetative growth of young plantlets, and reduces biomass production. This
study investigated the effects of priming with 1 mM S-methylmethionine
(SMM) on germination, leaf gas exchange, induced chlorophyll fluorescence,
photosynthetic pigment content and membrane damage through lipid peroxidation
by exposing canola (Brassica napus L. cv. Cindi) plants to moderate and
severe salt stress (induced by 60 mM and 120 mM NaCl) for
different periods. Priming with SMM alleviated the
reduction of net photosynthetic rate, the effective quantum efficiency and
efficiency of excitation energy capture by open photosystem II reaction centers,
chlorophylls to carotenoids ratio, enhanced water use efficiency and
contributed to reduction of oxidative membrane damage in fully developed young
leaves. Delay and inhibition of seed germination by salt stress were
significantly reduced by SMM, as well as the non-photochemical quenching of the
singlet excited state of chlorophyll a, suggesting a more efficient
protection against hyperosmotic stress, ionic toxicity and associated oxidative
stress in primed plants exposed to high salinity. This
first assay of SMM as a priming agent for canola plants under high salinity
contributes to a better understanding of the mode of action of this natural,
plant-derived bioactive compound and the optimization of canola cultivation
under the adverse growth conditions caused by salt stress. © 2021 Friends
Science Publishers
Keywords: Photosynthetic performance; Priming; Salt tolerance;
Stomatal conductance
Introduction
Priming of crop plants, at developmental stages
sensitive to adverse environmental conditions, proved to be a cost-effective
procedure which improves stress tolerance and enhances plant production without
any genetic manipulation (Tabasssum et al. 2017; Farooq et al. 2018).
Defense mechanisms may be accelerated or intensified by pre-treatment with
different substances which act as stressors, signaling molecules or
bioregulators (Yang and Lu 2005; Farooq et al. 2009, 2013, 2017, 2019; Sivaci
et al. 2014; Kaya et al. 2015). Even biological priming agents
(root-associated microbial communities, e.g., arbuscular mycorrhizal
fungi) were shown to be effective in crop stress management (Balestrini et
al. 2018; Rehman et al. 2018). Because short treatments with low
quantities of priming agents have multiple and long-lasting physiological
effects during hardening, the number of naturally occurring substances with
antistress potential is increasing, and novel agricultural practices are
developed in order to improve the bioproductive potential of several crop
plants in extended regions where climatic changes cause more severe abiotic
stress (Bajwa et al. 2018; Mahboob et al. 2019). For some
priming agents it was demonstrated that they induce transcriptional
modifications, post-translational protein modifications and epigenetic
modifications, which may confer prolonged protection during stress exposure.
Down-regulation of generation of reactive oxygen-nitrogen-sulfur species and
up-regulation of aquaporin and dehydrin expression levels were also attributed
to certain priming agents used to counteract hyperosmotic stress (Vannier et
al. 2015; Antoniou et al. 2016; Savvides et al. 2016).
Present understanding on how S-methylmethionine acts as a
priming agent in plants is very limited, and little information is available
about the influence of exogenous SMM on seed germination, leaf gas exchange,
photosynthetic parameters and membrane stability of canola exposed to salt
stress. Therefore, the objective of this study is to define effects of
pre-treatment with SMM on physiological processes in canola plants. This aims
to improve understanding of the mechanisms regarding the alleviation of
salinity stress and to apply priming with SMM for optimization of canola
growth, photosynthetic performance and salt stress tolerance. Our hypothesis is
that priming with SMM solution can enhance salt tolerance of canola by
attenuating negative effects of high salinity on photochemical processes of
photosynthesis, on gas exchange parameters and on other physiological traits
related to improved crop production and to acclimation to salt toxicity.
Materials and
Methods
Experimental details
The experiments
were carried out with canola (Brassica napus L. ssp. oleifera)
plants belonging to the “Cindi” cultivar. This was selected from ten frequently
grown cultivars (Avenir, Chalki, Chelsi, Cindi, Facti, Intense, Nodari, Kodiak,
Triangle and Tripti, the seeds being purchased from Caussade Semences, Alcedo
and KWS), based on previous studies which established that Cindi is the most
salt-sensitive. For germination, uniform sized healthy seeds, 100 in three
repetitions for each treatment setup, were selected and sterilized in 3% (w/v)
sodium hypochlorite solution for 10 min and washed twice with distilled water
(Abdolahi et al. 2012). For the other experiments, sterilized seeds were
pre-hydrated for 12 h with distilled water and germinated in Linhard vessels at
20°C. After one week, plantlets with similar size, ten for each experimental
variant (i.e., a total of 60 plants), were planted separately in pots
with perlite, watered regularly with half-strength Hoagland nutrient solution
(Hoagland and Arnon 1950) and grown for two weeks in an environmental chamber
(Sanyo MLR-351H) under a photosynthetically active photon flux density of 540 µmol m-2 s-1 for a
daily photoperiod of 14 h, at 22°C during the light period and 18°C in the dark
period, the relative air humidity being maintained at 60% (Zamani et al.
2010; Fiebelkorn and Rahman 2016). After two weeks of development, three series
of 10 plants were primed by spraying on their aboveground shoot (leaves and
stem) an aqueous solution of 1 mM S-methylmethionine. This concentration
was selected based on previous experiments, considering that quantities used
once as a pre-treatment are usually higher than those established for
continuous treatment during the experimental period (Paldi et al. 2014;
Fodorpataki et al. 2016). 12 h after the pre-treatment with SMM, several
series of ten plantlets each, primed and not primed, were exposed for 4 days to
moderate and severe salt stress, induced with 60 mM and 120 mM of
NaCl dissolved in the Hoagland nutrient solution used to irrigate the perlite
in the pots, once in two days at 8 a.m. Thus, six experimental variants were
set up, each with ten plantlets: the control provided with basic Hoagland
solution, a series pre-treated with 1 mM SMM but not exposed to
subsequent salt stress, a series exposed to 60 mM NaCl, one to 120 mM
NaCl and two series of ten plants each, primed with 1 mM SMM and then
submitted to 60 mM NaCl and 120 mM NaCl, respectively. Growth
conditions during the treatments were similar with those created for
development of seedlings after germination.
Measurements
Germination dynamics: Surface
sterilized seeds, three repetitions of 100 seeds each, were soaked for 12 h in
different aqueous solutions according to experimental variants: control and
non-primed lots were immersed in distilled water, while primed seeds were
covered by an aqueous solution of 1 mM S-methylmethionine. After 12 h,
all of the seed lots were put at equal distances from each other in Linhard
germination vessels, on double-layered filter paper thoroughly imbibed with
distilled water for control lots and for those receiving only SMM
pre-treatment, and with 60 mM and 120 mM NaCl solutions,
according to the two different salt treatments. Some salt-stressed seed lots
were not pre-treated with SMM, while the others were primed for 12 h with 1 mM
SMM. The seeds were kept at 20°C, 60% relative air humidity and 220 µmol m-2
s-1 photon flux density 10 h a day, for 8 days, the filter papers
being kept saturated with the treatment solutions. The germinated seeds (with
the radicle emerged through the seed tegument) were recorded every second day
during the morning hours.
Chlorophyll fluorescence: Conventional
and pulse amplification modulated parameters of induced chlorophyll
fluorescence were measured on the fourth emerging leaf of each plant, with an
FMS-2 type fluorometer (Hansatech). On dark-adapted leaves, just before the
onset of the light period, the ground fluorescence (F0) was measured
by illuminating the leaves with a dim red light flash (0.1 µmol m-2
s-1), while maximum fluorescence (Fm) was recorded during
a subsequent saturating light pulse (10000 µmol m-2 s-1
for 0.5 s). The leaves were then continuously illuminated with actinic light of
800 µmol m-2 s-1, in order to determine the steady
state fluorescence (Fs). A second saturating white light flash was
imposed to record the modulated maximum fluorescence in the light-adapted state
(Fm’), then the actinic light was turned off, and the modulated
minimal fluorescence in the light-adapted state (F0’) was measured
by illuminating the leaf with far-red light (30 µmol m-2 s-1
for 3 s). From the recorded values, the following parameters were calculated
and selected for interpretation: (1) the effective quantum efficiency of
photosystem II (PSII): ΦPSII = (Fm’ – Fs)/Fm’,
(2) the efficiency of excitation energy capture by open PSII reaction centers:
Fv’/Fm’ = (Fm’ – F0’)/Fm’,
and (3) the non-photochemical quenching of the singlet excited state of
chlorophyll a: NPQ = (Fm – Fm’)/Fm’
(Kooten and Snel 1990; Maxwell and Johnson 2000; Xia et al. 2004; Kalaji
et al. 2018).
Gas exchange parameters: Gas exchange
parameters were monitored at the start of salt treatment, on the second and on
the fourth day of exposure to salt stress, before noon, on the abaxial side of
the fourth fully expanded leaf from the base of stem, with a Ciras-2 type leaf
gas exchange meter (PP Systems). In the measurement chamber leaf temperature
was maintained at 22°C, CO2 concentration at 400 µmol mol-1,
the relative air humidity at 60% and light intensity at 540 µmol m-2
s-1 photosynthetic photon flux density. Net photosynthetic carbon
dioxide assimilation rate (Pn), stomatal conductance to water vapor (gs)
and transpiration rate (Tr) of leaves were measured, while the water use
efficiency (WUE) was calculated as the net photosynthetic carbon dioxide
assimilation rate to transpiration rate (Pn/Tr) ratio (Nieva et al.
1999; Zhang et al. 2009; Ceusters et al. 2019). Gas exchange and
chlorophyll fluorescence measurements were made, separately, on the same
portion of the leaf blades, between morning and noon.
Quantification of photosynthetic pigments: Photosynthetic
pigments were extracted from the fourth fully expanded leaf from the base of
the stem, from the same leaf blade region on which gas exchange and chlorophyll
fluorescence measurements were performed in vivo straight before pigment
determination, on the fourth day of salt exposure, around noon. Extracts were
obtained from 0.25 g of leaves (fresh weight) finely homogenized in 5 mL 80%
(v/v) acetone, then the supernatant resulting from centrifugation for 10 min at
4000g and 4°C was used for absorbance measurements, using a UV-Vis
spectrophotometer (Jasco). Concentrations of chlorophyll a, b and
carotenoids were calculated according to Wellburn (1994), then total
chlorophylls to total carotenoids ratio was computed.
Membrane lipid peroxidation assay: Oxidative
damage of membranes was evaluated on the basis of formation of malondialdehyde
(MDA) and other thiobarbituric acid-reactive substances (TBARS) due to
peroxidation of unsaturated fatty acids in membrane lipids (Baryla et al.
2000). 0.5 g fresh leaf samples, from the same leaves on which gas exchange
measurements, chlorophyll fluorescence determinations and photosynthetic
pigment quantifications were made, were homogenized with 5 mL of 0.1% (w/v)
trichloroacetic acid (TCA) solution in a pre-chilled mortar, the homogenate was
centrifuged at 15000 g for 15 min, then 2 mL of the supernatant was transferred
in a test tube and 4 mL of 10% (w/v) TCA with 0.5% (w/v) 2-thiobarbituric acid
(TBA) were added. The mixture was heated at 95°C for 30 min and then quickly
cooled in an ice bath. The cooled solution was centrifuged at 10000 g for 5
min, and the absorbance of the supernatant was measured at 532 nm and 600 nm.
The absorbance at 600 nm (due mainly to interference of anthocyanin pigments)
was deducted from the value obtained at 532 nm. MDA concentration of samples
was calculated using the extinction coefficient of 155 mM-1 cm-1 (Tang and
Newton 2005; Jambunathan 2010).
Fig.
1: Effects of NaCl and/or priming with
S-methylmethionine (SMM) on germination of canola seeds. Vertical bars
represent ± SE from means
Fig. 2:
Effective quantum efficiency of photosystem II (ΦPSII) in leaves of canola
plants exposed for four days to high salinity, with or without priming with
S-methylmethionine (SMM). C is the control without priming and salt treatment,
1 mM SMM refers to plants primed with
SMM but not exposed subsequently to NaCl stress. Bars represent ±SE from means
(n = 10), different letters above the columns indicate significant differences
at P < 0.05
Statistical analyses
All the
experiments were performed with ten plants for every treatment type, except for
germination, in which case experiments with 100 seeds for each treatment were
conducted in triplicate. Measurements of physiological parameters were repeated
three times. Data analysis was performed with the R statistical package (R Core
Team 2019), using the Shapiro-Wilk test for normality and Bartlett’s test for
homogeneity of variances. Data were represented as the mean ± standard error
(SE). The significant differences were determined by the one-way ANOVA test and
the post-hoc Tukey HSD test. Differences were considered significant at P
< 0.05.
Results
Germination
Priming for
12 h with 1 mM S-methylmethionine did not cause significant changes in
the dynamics of canola seed germination, but it considerably alleviated the
delaying and inhibitory effects of high salinity (Fig. 1). Control seed lots
had a germination percentage higher than 60% after two days, higher than 80%
after four days and a maximum of around 98% reached until the sixth day.
Pre-treatment with SMM did not result in significant changes, except for a very
moderate, but statistically significant decrease of germination percentage on
the fourth day, which recovered by the sixth day. Salt stress delayed
germination of most seeds and caused a lowered final germination percentage.
Under moderate salt stress (60 mM NaCl) less than 30% of seeds
germinated until the second day and around 60% germinated until the end of the
8 days period. When severe salt stress was applied with 120 mM NaCl, the
final germination percentage was lower than 40%, and during the first two days
less than 10% of seeds could germinate. These impairments caused by high
salinity were significantly counterbalanced by priming with SMM: germination
percentage of seeds exposed to 60 mM NaCl was increased from around 60%
to about 80% in six days, and on the second day more than 50% of seeds managed
to germinate (instead of an average of 26%). In the case of more severe salt
stress, the benefic influence of seed priming with SMM was even more
pronounced: the delay in germination was highly recovered between the second
and the fourth day (an average of 62% instead of 29% in non-primed seed lots),
and the final germination percentage was increased from 38 ± 2 to 74 ± 3%.
Photosynthetic performance
Effective
quantum efficiency of photosystem II (ΦPSII) was not influenced by priming
with 1 mM SMM in plants which were not exposed to salt stress.
ΦPSII of canola plants grown for four days in the presence of high
salinity was markedly reduced. This reduction was proportional with salt
concentration, and it was significantly alleviated by pre-treatment with 1 mM
SMM. In plants exposed to 60 mM NaCl, priming with SMM totally
annihilated the lowering effect of moderate salt stress, while in the plants
grown under a more severe salt stress (120 mM NaCl), pre-treatment
resulted in a partial compensation for the inhibitory effect (Fig. 2). The
efficiency of excitation energy capture by open PSII reaction centers (Fv’/Fm’)
of the plants treated with NaCl was reduced with time, this reduction being
more pronounced in the presence of 120 mM NaCl. Priming with
S-methylmethionine alleviated this reduction, and in the case of plants exposed
to 120 mM NaCl maintained a constant value of this photosynthetic
parameter. SMM had no significant influence on Fv’/Fm’ in plants not exposed to
high salinity (Fig. 3a). Non-photochemical chlorophyll fluorescence quenching
(NPQ) was shown to have different characteristics from the other photosynthetic
parameters determined in vivo in canola leaves, namely, it continuously
increased during the experiment in all plants exposed to salt stress,
irrespective of priming treatment. The NPQ of plants exposed to NaCl was
enhanced during the latter part of the experimental period, but this
enhancement was significantly attenuated in
SMM-primed plants, especially in the case of exposure to 120 mM NaCl.
Pre-treatment with SMM in unstressed plants had no influence on NPQ (Fig. 3b).
Net photosynthetic rate (Pn), measured through carbon dioxide uptake of leaves,
was reduced by salt treatment, the reduction being more pronounced in the first
half of the exposure period. This reduction was alleviated by priming with 1 mM
SMM at both NaCl concentrations, but the values remained under the ones
registered for control and only SMM pre-treated canola plants. On the fourth
day of salt treatment, the alleviation was more pronounced in plants exposed to
60 mM NaCl, in comparison with the ones grown in the presence of 120 mM
NaCl (Fig. 3c).
Photosynthetic pigment ratio
The ratio
between total chlorophylls (a + b) and total carotenoid pigment
content of fully developed canola leaf blades in the period of midday was
around 3.25 in control plants and pre-treatment with 1 mM
S-methylmethionine induced a moderate, but statistically significant increment
of this ratio. Salt treatment for four days reduced the chlorophylls to
carotenoids ratio proportionally with the salt concentration (to a value around
2.0 in the presence of 120 mM NaCl). This reduction was completely
offset by priming with SMM of salt-stressed plants, in which case the ratio
remained similar to control or was even higher in the primed plants exposed to
60 mM NaCl (Fig. 4).
Water use efficiency
Under non-salt
stress condition, spraying of an aqueous solution of 1 mM S-methylmethionine had no significant
Fig.
3: Effects of NaCl and/or priming with
S-methylmethionine (SMM) on the efficiency of excitation energy capture by open
photosystem II reaction centers (Fv’/Fm’) (a), on the non-photochemical
quenching of chlorophyll a fluorescence (NPQ) (b), and on the net
photosynthetic carbon dioxide assimilation rate (Pn) (c) in leaves of
canola plants. Bars represent ± SE from means (n = 10)
Fig.
4: Chlorophylls to carotenoids pigment ratio
in leaves of canola plants developed for four days under salinity, with or without
priming with S-methylmethionine (SMM)
effect on
water use efficiency of leaves during the subsequent four days, and maintained
its value around 0.002. Moderate salinity slightly increased WUE until the
second day of treatment and this value was maintained during the next two days.
Exposure to 120 mM NaCl resulted in a more pronounced increment of WUE
during the first two days, followed by a moderate, but significant decline
until the fourth day. Priming with SMM enhanced the
effect of salt treatment on the increment of water use efficiency at day 2
after starting the salt stress, but this enhancement was reduced on the fourth
day of treatment (Fig. 5).
Membrane lipid peroxidation
Thiobarbituric acid-reactive
substances (TBARS) content of canola leaves was not modified by pre-treatment
with 1 mM SMM, but it was significantly increased by salt stress, the
highest values being recorded in plants exposed to 120 mM NaCl. Priming
with SMM substantially reduced TBARS formation in salt stressed canola: in
plants exposed to 60 mM NaCl, SMM pre-treatment completely annihilated
the effects of moderate salinity on membrane lipid peroxidation, while in
plants treated with 120 mM NaCl, priming with SMM reduced peroxidative
membrane damage to the level of 60 mM NaCl-treated plants without
priming (Fig. 6).
Discussion
Germination
is a crucial developmental phase for the onset of new plants, during which
young plantlets are especially sensitive to adverse environmental conditions.
Salt stress slows down seed germination and results in erratic stand
establishment, leading to very low agricultural productivity (Abdolahi et al.
2012; Mahboob et al. 2019). The main inhibitory effect is due to the
osmotic component of salt stress, which impairs water absorption needed to
start the active growth and organogenesis. Seed priming with pre-soaking for 12
h in aqueous solution of 1 mM S-methylmethionine helped to maintain a
significantly higher germination percentage and reduced the time needed for
germination in salt stressed canola seed stands, thus improving salinity
tolerance through better emergence. This may be a simple and effective way to
overcome the negative impacts of high soil salinity on germination and on the
vigor of new plantlets (Tabasssum et al. 2017). Similar results were
reported for lettuce seeds, in which cold tolerance during germination and
seedling establishment was enhanced with pre-treatment using 0.25 mM and
2.0 mM SMM (Fodorpataki et al. 2019). Seed priming was also
achieved with a sequential pre-treatment with triacontanol and ascorbate,
resulting in improved germination index and reduced germination time of wheat
grains exposed to salt stress (Mahboob et al. 2019). They also
demonstrated that osmoprotectants’ accumulation was higher in primed seeds
exposed to salt stress, thus
osmoregulation may proceed
Fig.
5: Effects of two concentrations (60 mM and 120 mM) of NaCl and/or priming with S-methylmethionine (SMM) on the
water use efficiency (WUE) of canola leaves, calculated from stomatal gas exchange
parameters. Vertical bars represent ±SE from means (n = 10)
Fig. 6:
Membrane lipid peroxidation assayed with the amount of thiobarbituric
acid-reactive substances (TBARS) in leaves of canola plants exposed for four
days to salinity, with or without priming with S-methylmethionine (SMM). C –
control, FW – fresh weight
more
efficiently during germination in the presence of high salt concentrations. For
canola seeds, priming for 12 h with KH2PO4 (–0.625 MPa)
and CaCl2 (–1.25 MPa) resulted in vigor enhancement, internal
biological processes necessary for germination being intensified by priming
(Abdolahi et al. 2012). Seed soaking for 24 h before germination with
solutions containing thiamin (vitamin B1) promoted seedling growth
in maize cultivars exposed to salt stress (Kaya et al. 2015). All these
results suggest that seed priming may alleviate inhibitory effects of salt
stress during germination, pre-treatment being necessary only once and for a
short period of time, which makes this procedure cost-effective (Mahboob et
al. 2019).
Photosynthetic
efficiency decrease under NaCl indicates that moderate and heavy salt stress
during an exposure period of four days disturbs photochemical processes in PSII
and reduces the efficiency of light energy conversion into chemical energy in
leaves of young canola plants grown under constant light and temperature
regimes. Similar results were reported by Zhang et al. (2009) for
cucumber seedlings, as well as by Kalaji et al. (2018) for linden. The
fact that priming with S-methylmethionine had no effect on ΦPSII in plants
not exposed to high salinity, but significantly alleviated the reduction of
ΦPSII in salt-stressed canola plants (Fig. 2), suggests that this priming
agent does not have a direct influence on the functioning of PSII under normal
conditions, but it contributes to up-regulatory defense mechanisms which result
in an improved light use capacity under salt stress conditions. Demetriou et
al. (2007) reported that the protective role of polyamines in the
photosynthetic apparatus increases the efficiency of PSII photochemistry in
salt-stressed plants. Another photochemical quenching
parameter, the efficiency of excitation energy captured by PSII (Fv’/Fm’) of
canola plants exposed to salt stress was significantly reduced, this reduction
being intensified with time, especially in the case of treatment with 120 mM
NaCl (Fig. 3a). Priming with SMM alleviated this reduction, allowing the
salt-stressed plants to convert a higher proportion of the absorbed photons
into chemical energy which can be subsequently used for carbon dioxide
assimilation. Because the efficiency of PSII photochemistry in leaves of
salt-stressed canola plants primed with SMM is higher than without SMM
pre-treatment, one can conclude that SMM improved the photosynthetic capacity
of canola plants by increasing the level of photochemical efficiency of PSII
under salt stress conditions. Likely, when cucumber seedlings exposed for eight
days to 65 mM NaCl were simultaneously treated with 10 mM
putrescine, the exogenous polyamine supply alleviated the reduction of the
efficiency of excitation energy captured by open PSII (Zhang et al.
2009). The fact that exposure of canola plants to 120 mM NaCl
significantly increased NPQ (Fig. 3b) reflects that under the given light
intensity, salt stress reduced the photochemical efficiency of PSII, thus a
bigger proportion of the absorbed light energy was dissipated as heat in order
to avoid subsequent photooxidative damage to the photosynthetic apparatus
(Ruban 2016). Under conditions of moderate salt stress (60 mM NaCl) the
increase of NPQ is more pronounced after two days of salt treatment. In
SMM-primed canola plants the increment of NPQ was significantly lower than in
non-primed plants, suggesting the protective role of SMM against functional
damages induced by salt stress. Thus, S-methylmethionine enhanced salt
tolerance of canola by protecting the photosynthetic apparatus of the thylakoid
membranes due to photochemical quenching processes, the efficiency of this
preventive protection being associated with a moderated increase in heat dissipation.
A similar conclusion was drawn by Poulson et al. (2006), when they
primed with UV-B radiation Arabidopsis thaliana plants which were
subsequently exposed to drought stress.
Net
carbon dioxide uptake through stomatal gas exchange was shown to be related
directly to the rate of photosynthetic assimilation. Because stomatal closure
is a general defense mechanism under osmotic stress (caused mainly by drought,
extreme temperatures and high salinity), less carbon is assimilated under these
adverse conditions. This is the main reason why a greater part of the light
energy captured by photosynthetic pigments is not used photochemically, and has
to be dissipated or induces photooxidative damages which have to be repaired
with extra energy investment (Farooq et al. 2013; Ceusters et al.
2019). The present study showed that salt stress decreases net photosynthetic
carbon assimilation rate (Pn) proportionally with the NaCl concentration, and
mainly during the first days of exposure (Fig. 3c). This may be a result of
stomatal closure as a defense mechanism against osmotic stress induced by high
salinity. Stomatal closure impairs net carbon dioxide uptake, which results in
a decreased rate of photosynthetic assimilation (Ceusters et al. 2019).
The fact that Pn of plants without NaCl showed no influence by SMM, but priming
with SMM resulted in a more moderated reduction of this rate in salt-stress
plants, suggests that this substance enables canola plants to cope more
successfully with disturbances caused by high salinity in the photosynthetic
process. Because alleviation of salt stress by pre-treatment with 1 mM
SMM was less pronounced in the case of stomatal conductance for water vapor and
transpiration rate than in the case net carbon assimilation rate (data not
shown), one may conclude that SMM is involved in the improvement of
photosynthetic capacity in a higher extent by increasing the level of the
photochemical efficiency than by regulation of stomatal movements. This was
also found for putrescine in NaCl-stressed cucumber seedlings, but in the
latter case this polyamine had no effect at all on stomatal conductance and on
transpiration rate of leaves (Zhang et al. 2009). On the other hand,
priming with UV-B radiation had a much greater influence on transpiration rate
than on net carbon assimilation rate when Arabidopsis thaliana plants
were exposed to increasing light intensities (Poulson et al. 2006). In
wheat, high salinity (150 mM NaCl) caused a reduction in all gas
exchange parameters, while exogenous ascorbic acid induced a similar increase
in stomatal conductance for water vapor and in the carbon dioxide assimilation
rate of both salt-stressed and non-stressed plants (Farooq et al. 2013).
These results show that different priming agents may have disparate effects on
various parameters of stomatal gas exchange under several stress conditions.
The
fact that in absence of salt stress leaf spraying with SMM causes a moderate,
but statistically significant increase of the chlorophylls to carotenoids ratio
(due to a higher total chlorophyll content – data not shown) may be related to
the fact that, as an amino acid derivative, SMM stimulates the biosynthesis of
chlorophyll. Sadak et al. (2015) reported that foliar spraying of a
mixture of amino acids increased the chlorophyll content of faba bean leaves.
Zhang et al. (2009) reported an increment of chlorophyll content in
leaves of cucumber plants exposed to salt stress, with a tendency of reduction
at day 8 after the start of salt treatment. Intense UV-B radiation was also
shown to induce an increased photosynthetic pigment content of Arabidopsis
leaves. In several experiments, carotenoid pigment content increased when
stress factors impaired the use of light energy in carbon assimilation and a
higher amount of the absorbed light energy had to be dissipated in order to
prevent extensive photooxidative damage. This situation may occur under salt
stress, and the increment of photoprotective carotenoid pigment content may
explain the reduction of chlorophylls to carotenoids ratio in the canola leaves
exposed to high salinity. On the other hand, the fact that priming with 1 mM
SMM totally compensated for this influence of salt stress and in plants grown
in the presence of 60 mM NaCl even increased the value of this pigment
ratio above the value which was characteristic for the control plants, suggests
that SMM may participate in the acclimation of the photosynthetic apparatus to
high salinity conditions and may contribute to the protection of
thylakoid membranes against oxidative damage. This correlates with the results
which show that SMM pre-treatment reduces membrane lipid peroxidation in plants
exposed to salinity stress. A similar role of putrescine was demonstrated in
cucumber seedlings exposed to salt stress induced by a treatment for 8 days
with 65 mM NaCl (Zhang et al. 2009).
Pre-treatment
with 1 mM SMM had no influence on WUE, while in two days after the onset
of salt stress a significant increase of water use efficiency indicates that
the osmotic component of high salinity induces regulation of stomatal
conductance to water vapor and of carbon dioxide assimilation, which results in
increased drought tolerance. Transpiration rate is lowered by salt stress more
intensely than net carbon dioxide assimilation, thus water use efficiency
increases. This increment is intensified if salt-stressed plants were primed
with SMM, the enhancement being reduced on the fourth day of salt treatment.
Because SMM alleviated the inhibitory effect of salt stress on net
photosynthetic carbon assimilation, but exerted a weaker influence on
transpiration rate significantly reduced by salt stress due to stomatal closure
(data not shown), one can conclude that S-methylmethionine affects the
photosynthetic carbon assimilation rather than stomatal movements. Partly
similar results were obtained when salt-stressed cucumber seedlings treated
with 10 mM putrescine exhibited an enhanced water use efficiency at day
1 after the beginning of salt treatment, but during the next days the
enhancement was gradually reduced (Zhang et al. 2009). This was related
with the fact that putrescine moderated the reduction of net photosynthetic
rate by salt stress but had no significant influence on stomatal conductance
and transpiration rate. In wheat, salt stress induced with 150 mM NaCl
decreased water use efficiency because of a more intense reduction of carbon
dioxide assimilation rate than that of transpiration rate, while priming with
100 mg L-1 ascorbic acid (vitamin C, applied to the rooting medium)
caused an increase in WUE of non-stressed plants, but had no significant
influence on WUE of salt-stressed plants (Farooq et al. 2013).
In
the present study it was found that exogenous application of 1 mM SMM
before the exposure of young canola plants to moderate and severe salinity
stress mitigated the effect of salt stress on membrane damage reflected by
significant accumulation of TBARS. This can be associated with reduced
generation of reactive oxygen species in SMM-treated canola plants subsequently
exposed to salt stress. It is suggested that increase in antioxidative
activities in salt-stressed plants is an additional burden on metabolism which
may be reduced by priming with SMM. Similar results were reported by Kaya et
al. (2015) for salt-stressed maize cultivars, when exogenous application of
thiamine reduced hydrogen peroxide and malondialdehyde formation by modulating
the cellular redox status and the activity of antioxidant enzymes, resulting in
a reduced metabolic cost of stress tolerance. Pre-treatment with 100 mM
ascorbic acid also reduced membrane lipid peroxidation in pepino plantlets
exposed to chilling stress, and its beneficial influence on stress tolerance
was related to the fact that it modulated the activities of antioxidant enzymes
involved in membrane protection (Sivaci et al. 2014). The current
results support the idea that priming with millimolar amounts of
S-methylmethionine as foliar spray may efficiently prevent oxidative membrane
damage induced by salt stress in the leaves of canola plants. However, the role
of exogenously applied SMM in the antioxidative defense system of plants yet
needs to be elucidated.
Conclusion
Priming with S-methylmethionine can be effectively used
to enhance salinity tolerance of canola during its early developmental stages.
It compensates for the delayed germination under salt stress, restores
photosynthetic performance under moderate salt stress and alleviates its
reduction by severe salt stress. Reduction of chlorophylls to carotenoids ratio
and increment of membrane lipid peroxidation in leaves are annihilated by
pre-treatment with SMM as a foliar spray, while water use efficiency is
improved under high salinity. The current results open a perspective for
metabolic engineering aiming an enhanced production of SMM by the canola plant
itself, to achieve an increased production under adverse cultivation
conditions. From a crop improvement perspective, modulation of SMM metabolism
using its exogenous application may also be an innovative way to improve
abiotic stress tolerance in several crop plants.
Acknowledgements
We thank for the support given to Ph.D. student Katalin
Molnar by the doctoral school of the University of Agricultural Sciences and
Veterinary Medicine in Cluj-Napoca, and to M.Sc. student Bernat Tompa by the
Institute of Advanced Studies in Science and Technology of the “Babes-Bolyai”
University in Cluj-Napoca, Romania.
Author Contributions
Laszlo
Fodorpataki: planning and supervision of the work, methodology and editing.
Katalin Molnar: methodology and data analysis. Bernat Tompa: methodology and
data analysis. Csaba Bartha: methodology, data analysis, review and editing.
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