Introduction
Salt stress is one of the major limiting abiotic
factors for crop production globally. According to Food and Agriculture
Organization (FAO), over 830 million ha of land all over the world suffers from
soil salinization (Liu et al. 2019). Due to high salinity, plants
growing in
salinization suffer from serious ion toxicity (Huang et al. 2017;
Al-Farsi et al. 2020) and continuous osmotic stress (Farooq et al.
2017; Li et al. 2017). The salt stress inhibit growth (Li
et al. 2017), photosynthesis (Nishimura et al. 2011;
Xu et al. 2019), chlorophyll fluorescence (Dadkhah 2015; Xu et al.
2019), nutrient uptake and metabolism (Pattanagul and Thitisaksakul 2008; Zhao et
al. 2014), ionic imbalance (Zhu 2003;
Vijayalakshmi et al. 2014; Farooq et al. 2015) and
finally suppress the yield of crop plants (Wang et al. 2010, 2016). This
makes it imperative to improve crop plants yield under salt stress conditions.
As important grain crops in China,
rice plays an important role in grain production but sensitive to salt stress
as well (Grattan et al. 2002). Recently, the planting area of rice in
Jilin Province has been greatly increased compared with other crops, but the
probability of salt damage to rice has also been greatly increased (Wang et
al. 2010). Studies have shown that salt stress inhibit rice seed
germination (Munns 2002; Wang et al. 2015), delays the growth and
development of rice plants (Koca et al. 2007; Abbasi et al. 2015)
and eventually lead to fearful decline of grain yield (Bhantana and Lazarovitch
2010; Bybordi 2010). In addition, salt stress disrupts physiological metabolism
of rice plants by reducing photosynthesis (Nishimura et al. 2011; Liu et
al. 2014), reduce absorption of water and nutrients (Golldack et
al.
2014), damage living cells (Moradi and Ismail 2007; Zhang et al.
2017), destroy ROS-scavenging systems (Moradi and Ismail 2007) and ion balance
(Summart et al. 2010; Wang et al. 2012). Therefore, to cope with
the most impelling challenge of providing enough food and effectively utilize the
salinization soils in Jilin Province, it is still essential to continue
in-depth studies on how rice plants response to salt stress at different growth
stages and seeking effective approaches for the improvement of crop plants to
salt stress.
There are numerous approaches to
enhance crop plant resistance to salt stress including breeding salt tolerant
crop varieties (Hanin et al. 2016), appropriate nutrition management
(Siddiqui et al. 2010) and exogenous application of chemical substances
(Wei et al. 2017; Liu et al. 2019). Among these, appropriate
nitrogen levels promote the development and growth of crop plants, and could
enhance resistance to environmental stress
(Hoai et al. 2003; Gamett et al. 2009; Siddiqui et
al. 2010). Previous researches have shown that the nitrogen levels affect the growth and development of cotton
under salt stress at the later growth stages (Chen et al. 2010). In
sunflower, low nitrogen concentration was more favorable to
enhance its resistance to salt stress, while high nitrogen concentration more
harmful to its root system (Ashraf
and Sultana 2000). In rice, increasing nitrogen application caused decrease in
root dry mass of rice under 50 and 100 mmol/L of salt stress (Abdelgadir et
al. 2005). Our previous study showed that reducing nitrogen
application was helpful of rice plants to alleviate the salt-induced decline of
leaf photosynthesis and damages to photosynthetic apparatus after booting stage (Xu et al. 2019) and
lower nitrogen levels contributed to improve salt tolerance of rice at later
growth stage. However, how different nitrogen levels
influenced the growth and physiological response of rice plants under salt
stress conditions remains unclear.
This study aimed to explore how
different nitrogen levels influenced the plants growth, physiological
metabolism and gene expression under salt stress conditions. This study also
showed that lower nitrogen levels contribute to improve rice plant growth,
increase accumulation of osmolytes and
to mitigate plant damage and ion toxicity under salt stress conditions
to improve salt tolerance after booting stage. This study would provide
theoretical basis and scientific proof for the rice production in the
salinization soil area of Jilin Province.
Materials and methods
Plant materials
Two conventional japonica rice (Oryza sativa subsp. japonica)
varieties grown in northern China with different levels of salt tolerance:
‘Changbai 9’ (moderately tolerant to salt stress) and ‘Jinongda 19’ (sensitive
to salt stress) were used in this study. Both the two varieties were
medium-early maturating, and selected from 112 rice varieties grown in Jilin
Province (Xu et al. 2013).
Plant growth
conditions and salt-stress treatments
Rice seeds were sterilized using
75% (v/v) alcohol for 5 min and then rinsed using
deionized water. The rice germination was carried out in a culture dish.
The plants were transplanted into buckets (with the diameter of 35 cm and
height of 45 cm) with 5 cm spacing at plant height greater than 10 cm. Rice
roots were fixed with absorbent cotton and inserted into a perforated black
plastic foam plate. Eight uniform rice plants were put into buckets of improved
IRRI Yoshida nutrient solution with different nitrogen levels. The nitrogen
levels used were 1/4, 1/2, 1, 2 and 4 times of the normal nitrogen content (1N)
using ammonium nitrate. The normal nitrogen level in the nutrient solution was 0.715 mmol/L. The different nitrogen
concentration with 4, 2, 1/2, and 1/4 times of the normal level in the nutrient
solution were 2.86, 1.43, 0.3575 and 0.17875 mmol/L, respectively. The nutrient
solution was replaced once every seven days and pH value was adjusted to 5.1 ± 0.2.
Rice plants were grown in nutrient
solution with different nitrogen levels and salt stress was imposed at tillering,
booting and heading stages, respectively. The NaCl solution of 80 mM
were put into each nitrogen levels (1/4N, 1/2N, 1N, 2N and 4N), and the normal
nitrogen level without salt stress was taken as control (CK). All rice plants
were grown in a controlled growth chamber with growing temperature of 25 ±0.5°C day/20 ± 0.5°C night, and light intensity of 12 h
photoperiods with 350 μmol
photons m-2 s-1. Rice leaves and roots of each treatment were sampled to measure the
biomass accumulation and physiology indices after 7 d of salt stress, and gene
expression was after 3 d of salt stress, respectively.
Measurement of
withered leaf rate
The withered leaf rate was
investigated at the tillering, booting and heading stages, respectively. Rice
leaf was regarded as withered if the whole
leaf was dry and brown and expressed as withered leaf rate represented by the
proportion of withered leaves (Wei et al. 2017; Liu et al. 2019).
Measurement of
Biomass accumulation
Three plants were selected for
biomass determination. After decomposition, the fresh samples were fixed for 10
min at 105°C in a drying oven. The dry matter was
determined after the temperature was adjusted to 80°C for drying for 48 h to a
constant weight.
Measurement of
soluble sugars and proline content
A 0.1 g dry grounded leaf sample
was placed into a 10 mL centrifuge tube with a cover, and distilled water was
added until the tube was 2/3 full. The water extraction liquid was obtained by
water bath pot distillation for 40 min. The water extraction liquid was used
for the determination of soluble sugars and proline content. The soluble sugars
were detected with anthrone colorimetry according to Xu et al. (2013). 1
mL of the sample with 5 mL of the anthrone reagent was used. The sample was
boiled in a water bath for 10 min and absorbance was measured at the wavelength
of 620 nm after cooling. The proline was detected by the sulfonyl salicylic
acid method. For which, 2 mL extracting solution was taken and added 2 mL of
the glacial acetic acid and 2 mL of acid indenone reagent. The solution mixture
was boiled for 30 min, and then 4 mL toluene was added after
cooling. The supernatant was centrifuged for 5 min and after shake for 30
seconds. Toluene was used as the reference for color comparison at 520 nm
wavelength on spectrophotometer.
Measurement of
Malondialdehyde content and membrane injury
The MDA content was determined
using the thiobarbituric acid reaction method (Heath and Packer 1968), with
some modifications (Liu et al. 2019). The fresh sample (0.1 g) was
smashed and then homogenized in the phosphate buffer (pH 7.8) using a ball-mil,
and then centrifuged for 15 min. Subsequently, 400 μL of the
supernatant was taken, and then mixed with 1 mL of the thiobarbituric acid.
After which, the mixture was heated at 100°C for 30 min, and then centrifuged
after cooled sufficiently. The absorbance of the supernatant was measured at
532, 600, and 450 nm using a spectrophotometer to determine the MDA content of
rice leaves by the formula: 6.45× (A532 – A600) - 0.56 ×
A450.
The membrane injury (MI) was
measured using the electrolyte leakage method according to the description (Liu
et al. 2019). The MI of rice leaves was evaluated using the formula (%)
= EC1/ EC2 ×100%. The EC1
and EC2 was represented for the electrical conduction of leaves
without or with boiled, respectively.
Measurement of
the sodium and potassium
Content of sodium and potassium
were measured according the description by Wei et al. (2015). The dried
samples were cut with a scissor to 5–10 mm pieces and digested completely with
HNO3 and HClO4 (v/v = 2:1) mixture and diluted to 50 mL.
The Na+ and K+ concentrations were determined by flame
emission spectrometry.
RNA isolation
and quantitative real-time PCR (qRT-PCR)
The RNA isolation, reverse transcription, and quantitative real-time PCR
(qRT-PCR) of every sample was executed (Liu et al. 2019). Using TRIzol
reagent (TaKaRa Bio Tokyo, Japan) for the extraction of total RNA of rice
leaves, and using M-MLV reverse transcriptase (Thermo, Carlsbad, C.A., U.S.A.)
for the synthesis of first-strand cDNA. The transcriptional expression of all
genes was determined by quantitative real-time PCR (qRT-PCR). The
transcriptional expression levels of each gene were calculated using the 2-△△CT method (Livak and Schmittgen 2001).
And the reaction mixture and procedure of qRT-PCR were performed as described
by Liu et al. (2019). The housekeeping gene β-actin (GenBank ID:
X15865.1) was used as an internal standard. The gene-specific primer pairs designed by Primer 5.0 software, and used for qRT-PCR as
follows:
OsACT1: 5′-TTCCAGCCTTCCTTCATA-3′ and 5′-AACGATGTTGCCATATAGAT-3′;
OsP5CS1: 5′-TGTGTACCAACGCGCTATGT-3′ and
5′-TATATGCATCCACGGCGATA-3′;
OsP5CS2: 5′-GTGGCTTGTGAAGGAGCTGT-3′ and
5′-TTTGACATGCTTTCGTGCTC-3′;
OsPDH1: 5′-GCTACTGGGACTTGGGAGTG-3′ and
5′-TCGATTGATACACCAATGTCTG-3′;
OsP5CDH: 5′-TCTGAATAATTTGCCCCGTCT-3′ and
5′-CACAACCATTTCCTGCCTTT-3′;
OsNAC4: 5′-TGGATGGAGCAAGAAAAAGG-3′ and
5′-CCACCACATTTGCAGAATCA-3′;
OsBI1: 5′-CTACATCAAGCACGCACTC-3′
and 5′-ACCTCTTCTTCCTCTTCTTCTC-3′;
OsHKT1: 5′-ACACCCAATATTATTCCTCTTAA-3′
and 5′- CGGGAATACGCTAAAGG-3′;
OsAKT1: 5′-AGAGATCCTTGATTCACTGCC-3′ and
5′-TCTACTAACTCCACACTACCAG-3′
Statistical
analyses
Each treatment was replicated
thrice in a controlled growth chamber. The data software S.P.S.S. 21.0 (I.B.M.
Corp., Armonk, N.Y.) was used for the statistical analyses. Based on the
results of ANOVA, a Duncan’s multiple range test (DMRT) was used for the mean
comparison. And the significance level among different treatments was P < 0.05.
Results
Effects on leaf
withering
Lower nitrogen levels reduced leaf withering under salt stress as
shown by lower withered rates of leaves (Fig. 1C, 1D and 1E). The withering
rates of leaves of 1/2N were the minimum of all the nitrogen levels under salt
stress at booting (Fig. 1A and B) and heading stages, but the minimum leaves
withered rates was for 1N at tillering stage (Fig. 1C, 1D, and 1E). Changbai 9
was more tolerant than Jinongda 19 to salt stress according to the growth
condition under salt stress (Fig 1A, 1B).
Effects on
aboveground and root biomass
Salt stress caused a significant
decrease in biomass accumulation to both two rice cultivars as shown by decrease
of the aboveground and roots dry weight compared with the control (Fig. 2). The
decline in biomass of the salt-tolerant
rice variety Changbai 9 was lower than salt-sensitive rice variety Jinongda 19.
Compared with the control, the aboveground dry weight of plants exhibited a
lower magnitude of decline for the 1N, 1N, and 1/2N treatment at the tillering,
booting and heading stages, respectively (Fig. 2A, 2B, and 2C). The decline of
root biomass salinity-induced under different nitrogen levels was similar to
above-ground part (Fig. 2). Compared with control, the dry weight of the roots
exhibited a lower magnitude of decline in the 1N, 1N, and 1/2N treatments at
the tillering, booting, and heading stages (Fig. 2D, 2E, and 2F). The shoot and
root dry weight of 1N and 1/2N plants were not statistically different at any
stages.
Osmolytes
accumulation during different growth stages
%20769-776_files/image002.png)
Fig. 1: Effect of salt stress on seedling growth under different nitrogen levels. Photographs of seedling growth (A, B) after 7 d of salt
stress at booting stage. Rate of withered leaves (C, D, E) were counted after 7 d of salt
stress. Values are means ± SD, n=3.
Different letters on the column represent significant difference (P < 0.05) between each treatment of
the same rice variety based on Duncan’s test
%20769-776_files/image004.jpg)
Fig. 2: Effect of salt stress on seedlings biomass
accumulation under different nitrogen levels. Aboveground biomass (A, B,
C) and root biomass (D, E,
F) after 7 d of salt stress
A remarkable accumulation of
proline and soluble sugar was observed under salt stress conditions (Fig. 3A–F),
and the accumulation of osmolytes in Changbai 9 were more than Jinongda 19
(Fig. 3). Compared with control, the increase of osmolytes in the 1N treatment
was the greatest at tillering stage, and 1/2N at booting and heading stages
(Fig. 3), respectively. Therefore, it is speculated that 1N, 1/2N, and 1/2N
treatments supply the optimal nitrogen fertilization levels for rice at the
tillering, booting, and heading stage, respectively.
Effects on lipid
peroxidization of cytomembrane and membrane damage
Salt stress significantly caused
peroxidization of cytomembrane and cell damage by the increase of malondialdehyde (MDA) and membrane injury (MI) under
salt stress conditions (Fig. 4). Compared with control, the MDA content
exhibited a lower magnitude of increase in the 1N, 1/2N, and 1/2N treatment at
tillering, booting, and heading stages, respectively (Fig. 4A–C). Consistently,
the decrease of MI exhibited the highest in 1N, 1/2N, and 1/2N at tillering,
booting and heading stages (Fig. 4D–F), respectively. Therefore, lower nitrogen
levels mitigated salinity-induced peroxidization of cytomembrane and cell
damage of rice under salt stress conditions at the later growth stages.
Ion toxicity of
rice at later growth stages
The Na+ content
significantly induced and decreased in K+ in response to salt stress
treatment. This salinity-induced Na+ accumulation was significantly
suppressed by lower nitrogen levels application at tillering, booting and
heading stages (Fig. 5A, 5B, and 5C). In contrast, the K+ content
under salt stress was significantly increased by lower nitrogen levels
application as well (Fig. 5D, 5E, and 5F). Furthermore, the 1/2N treatment
showed the minimum Na+ and maximum K+ contents of all the
treatments (Fig. 5).
Lower nitrogen
levels superinduced stress tolerance-related genes
%20769-776_files/image006.jpg)
Fig. 3: Effect of salt stress on soluble sugar and proline
content of leaves under different nitrogen levels. Content of soluble sugar (A, B,
C) and proline
(D, E, F) after 7 d of salt
stress
%20769-776_files/image008.jpg)
Fig. 4: Effect of salt stress on MDA content and MI under
different nitrogen levels. MDA content (A,
B, C) and MI (D, E, F)
after 7 d of salt stress
%20769-776_files/image010.jpg)
Fig. 5: Effect of salt stress on Na+, K+ content of shoot
under different nitrogen levels. Na+ (A, B, C) and K+ (D, E,
F) contents after 7 d of salt stress
%20769-776_files/image012.jpg)
Fig. 6: Effect of salt stress on relative expression
levels of proline-related genes OsP5CS1 (A), OsP5CR (B), OsPDH1 (C), OsP5CDH
(D) and stress tolerance genes OsNAC4 (E), OsBI1 (F), OsHKT1 (G), OsAKT1
(H) under different nitrogen levels
at heading stage
The two proline biosynthesis genes
(OsP5CS1 and OsP5CR) were induced by salt stress, and the
induction levels were much higher in 1/2N treatment. But the two proline catabolism genes (OsPDH1 and OsP5CDH)
were much suppressed by the 1/2N treatment (Fig. 6C–D). These results were
consistent with the proline content (Fig. 3D, 3E, and 3F), which indicated that
lower nitrogen levels contributed to accelerate synthesis of proline and
suppressed catabiosis under salt stress. In
addition, the expression levels of OsNAC4,
a cell death-related gene, were significantly suppressed by lower nitrogen
levels and the downregulation of the cell death suppressor, OsBI1 was alleviated in the 1/2N
treatment (Fig. 6C, 6D). The expression levels of Na+ transport gene,
OsHKT1 and the K+ channel
regulation gene, OsAKT1 showed
upregulation in response to lower nitrogen levels under salt stress conditions
(Fig. 6E, 6F), which indicated that the transfer efficiency of Na+ and
K+ were better than higher nitrogen levels.
Discussion
Salinity is one of most severe
abiotic stresses that limit crop growth and yield production, and plants
response is complex. Hence, many measures of improving rice salt tolerance
could be applied according to the response between plants and salt stress.
Appropriate nutrient management not only promote crop
plants growth effectively, but also improve resistance
to various environmental stresses (Soussi et al. 1998; Silveira et al.
2001). Salt stress has a stronger effect on nitrogen metabolism in rice and its
regulation plays important role to cope various stresses conditions (Läuchli and Lüttge 2002;). Medium or lower nitrogen levels application for
enhanced tolerance to salt stress have been reported in cowpea, sunflower, and
soybean (Silveira et al. 2001; Ashraf and Orooj 2005; Hamayun et al.
2010). Our previous study showed that reducing nitrogen levels enhanced salt
tolerance of rice by improving photosynthesis efficiency and protecting
photosynthetic structures after booting stage (Xu et al. 2019). However,
what the physiological and transcriptional expression response of rice plants
under different nitrogen levels to salt stress have remained unknown. Results
of the present study showed that lower nitrogen levels significantly improved
rice plants growth (Fig. 1, 2) and physiological metabolism by increasing
accumulation of osmolytes (Fig. 3, and Fig. 6A–6D), and mitigating cell damage
(Fig. 4, and Fig. 6E, 6F) and ion toxicity (Fig. 5; Fig. 6G–H) at salinity
after booting stage.
Plants accumulate osmotic
regulatory substances to increase the mass concentration of cell fluid and
reduce osmotic potential to resist salt damage (Redillas et al. 2012).
Proline and soluble sugar play important role in the osmotic adjustment of
rice. This study showed that salt stress induced accumulation of osmolytes in
rice, and compared with the control, the largest increase of osmolytes were in
the 1N, 1/2N, and 1/2N treatments at the tillering, booting, and heading
stages, respectively (Fig. 3). After booting and heading stages, 50% percent of
the nitrogen level in the nutrient solution could induce more osmolytes
accumulation under salt stress, which were more beneficial for rice to cope
with salt stress (Hoai et al. 2003). The increase of proline was due to
upregulation of the proline biosynthesis genes (Fig. 6A–B) and suppression of proline
metabolism genes (Fig. 6C–D) by lower nitrogen
levels. These results suggested that lower nitrogen levels contributed to
promote biosynthesis of osmolytes for enhanced capacity of osmotic adjustment
in rice to resist salinity-induced osmotic stress (Summart et al. 2010).
MDA is one of the products of
membrane lipid peroxidation, and thus MDA and MI are both important indicators
by which to judge the integrity of the membrane and
the degree of cell damage under stress conditions (Tang et al. 2007; Liu
et al. 2019). In this study, lower nitrogen levels decreased MDA content
(Fig. 4A–C) and MI (Fig. 4D–F) of rice, which indicated that milder salinity-induced membrane injury, was exhibited under lower nitrogen levels after booting
stage. To the cell damage, lower nitrogen levels down-regulated expression levels of OsNAC4 (Fig. 6E) and up-regulated of OsBI1 (Fig. 6F), which
indicated that lower nitrogen levels mitigated salinity-induced cell
death in rice. These results indicated that lower nitrogen levels application
for rice plants could enhance salt tolerance by protecting cell construction
more efficiently than high nitrogen levels.
High ion toxicity was an important
damage mechanism by salt stress to plants, such as Na+ and Cl-,
and plants with greater tolerance to salt stress
generally showed lower uptake of Na+ (Yang et al. 2008; Lv et
al. 2013), while some ions contribute to improve rice salt tolerance, such
as Ca2+ and K+. Exogenous Ca2+ could improve
photosynthesis efficiency and increase accumulation of osmolytes for the
improvement of salt tolerance in rice (Zhu et al. 2004, 2005). Increase
of K+ could replace sodium ions of rice plants to improve salt
tolerance of rice (Peng et al. 2004). Thus, rice with greater tolerance
to salt stress showed lower Na+/K+ (Lv et al.
2013). In present study, Na+ content was significant lower and K+
contents was higher in the lower nitrogen levels (in the 1/2N treatment) at the
three stages (Fig. 5), indicating that lower uptake of Na+ and
disturbance of K+ by salt stress, which improved tolerance to salt
stress of rice. In addition, rice plants could keep efficient activation of
Na-K transportation under lower nitrogen levels by significant upregulation of OsHKT1 and OsAKT1 (Fig. 6G–H), when salt stress was approached. These results
suggested that keeping the ion balance in rice for mitigating salinity-induced
high ion toxicity was an important pathway of lower nitrogen levels help rice
to cope with salt stress conditions.
Nitrogen fertilizers play an
important role in enabling crops growth normally and cope with environmental
stress conditions. But higher nitrogen levels do not contribute to mitigate
salinity-induced growth inhibition and damage to crop plants (Papadopoulos and
Rendig 1983; Abdelgadir et al. 2005). The similar conclusion was showed
in the saline-alkaline field conditions where higher nitrogen fertilizer
aggravated the saline-alkaline degree of soil and resisted the transfer of
nitrogen element, which suppressed growth of plants (Qi et al. 2014; Du et
al. 2015). In the present study, the best nitrogen level was 1N treatment
to counteract salinity-induced damage to rice at tillering stage, which
indicated that enough nitrogen application could contribute to improve growth
and salt tolerance of rice plants at the vegetative period of rice. While at
booting and heading stages, the best nitrogen level was 1/2N treatment (50%
percent of the normal N), indicating that moderate reducing nitrogen levels
enhanced salt tolerance of rice by improving some physiological traits from the reproductive stage. These results suggested that
reducing nitrogen and improving nitrogen use efficiency will be a vital pathway
for enhanced salt tolerance of rice plants from the reproductive stage, and how
to improve the nitrogen use efficiency under stress conditions will be an
interesting research topic.
Conclusion
Salt stress induced wilting and
growth inhibition, aggravated lipid peroxidation and cell damage, and increased
Na+/K+ ratio. Application of lower nitrogen levels
improve rice salt tolerance by increasing osmolytes accumulation, mitigating
membrane damage, keeping the balance of Na+ and K+, as
well as upregulating stress tolerance-related genes to rescue rice plants for adapting to salt stress after
booting stages.
Acknowledgements
The authors would like to thank the
National Key Research and Development Program of China for its financial
support (Project Numbers: 2018YFD0300205); the Technology Development Program
of Jilin Province in China (Project Numbers: 20170203003NY); the Doctorial
Scientific Research Fund of Yichun University (Project Numbers: 210-3360119017)
for their financial support.
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