Root Architectural and Physiological Responses in Contrasting Rice Genotypes to
Saline-Alkaline Stress
Xiaolong Liu1†,
Chen Xu2†, Hongtao
Yang1, Peipei Su1, Qin Shao1, Na Chen1,
Liannan Lin1, Zhian Zhang3 and
Hongjun Wang2*
1College of Life Sciences and Resources
and Environment, Yichun University, Yichun, P. R. China
2Institute of Agricultural Resources and Environment Research, Jilin
Academy of Agriculture Sciences, Changchun, P. R. China
3Department of Agronomy, Jilin Agriculture University, Changchun, P. R. China
*For correspondence: whj603@126.com
†Contributed equally to this work and
are co-first authors
Received 03 March 2021;
Accepted 18 June 2021; Published 18 September 2021
Saline-alkaline
(SA) stress suppress rice growth by severely inhibiting root growth and
damaging root system. This study investigated the main
limiting factor for root growth in rice under SA stress. Four conventional
japonica rice with different saline-alkaline tolerance, Dongdao-4 (D4),
Changbai-9 (C9), Jinongda-19 (J19) and Nipponbare (NB) were used in this study.
Two-week-old rice seedlings were grown under different types of SA stress
simulated by 120 mM NaCl, 60 mM Na2SO4, 30 mM NaHCO3 and 15 mM Na2CO3,
respectively. Root growth indices including total root length (TRL), total root
surface area (RSA), total root volume (TRV), average root diameter (ARD) and
root numbers (RN), and some physiological traits i.e., Na+, K+, proline, soluble sugar,
superoxide anions (O2•-) and hydrogen peroxide (H2O2)
contents were measured in roots. Results showed that all root growth indices
significantly decreased by SA stress. The TRL, RSA, TRV and RN of rice
seedlings suppressed severely by Na2CO3 stress, but the
ARD suppressed severely by NaCl stress. The SA stress induced overaccumulation
of Na+, proline, soluble sugar, O2·- and H2O2
in rice roots. More accumulation of Na+, proline and soluble sugar
was observed in NaCl treatment, but Na2CO3 treatment
induced more accumulation of O2•-
and H2O2. Root growth
indices showed significant correlations to O2•-, H2O2,
Na+, proline and soluble sugar contents under SA stress. Root growth
and physiological responses of saline-alkaline tolerant cultivars (D4 and C9)
were more superior than sensitive cultivars (J19 and NB). These results suggested that suppression of root growth was a combined effect of osmotic stress, ion toxicity and
oxidative stress induced by SA stress. Oxidative stress induced by
overaccumulation of O2•- and H2O2
resulted in severe damage to root by inhibiting its elongation and growth of
new tips. © 2021 Friends Science Publishers
Keywords: Physiological traits; Rice (Oryza sativa L.); Root growth indices; Salt stress;
Alkali stress
Introduction
There are over 830 million ha of saline-alkaline soils
all over the world (FAO 2016), which result in severe inhibition for growth and
yield formation to crops grown in these types of soils. Soil salinization and
alkalization is commonly divided into neutral salts which refers to
NaCl and Na2SO4, and carbonates which refers to NaHCO3
and Na2CO3 (Yang et al. 2007; Lv et al.
2013). Saline stress includes the character of high salinity and high osmotic
pressure, and generally plants experience osmotic and high ion
toxicity under these stress type (Liu et al. 2019, 2020).
While, alkaline stress induces high pH stress in addition to salt stress, which
damage plants directly (Zhang et al. 2017; Liu et al. 2019).
Consequently, plants grown in saline-alkaline soils suffer from osmotic stress,
ion toxicity and high pH stress together.
Roots are primarily exposed to soil or water solution
and suffer from various stress conditions (Koevoets et al.
2016; Kaashyap et al.
2018). Root also absorbs water and various nutrients from soil for
plants growth. Hence, the morphological characteristics such as root length,
surface and root hairs, as well as the physiological traits play vital role in
determining plant growth and yield production (Ghosh and Xu 2014). Plants with higher root length could acquire
water and nutrition from deeper soil beneficial for plants to adapt the drought
condition (Kim et al. 2020). Roots with smaller diameter and higher root
length increase the surface area of root in contact with the water in soils,
which enhance the volume of soil with water (Hernández et al.
2010; Comas et al. 2012).
Roots of diameter 0.5‒2.0 mm are “fine” and decrease of diameter contribute to the enhance
access to water in soil and production under water stress (Zobel and Waisel 2010; Wasson et al. 2012). Root hairs and new root tips
are the key indicators determining root continuous growth and vital for the
uptake to water and nutrition in soil, responsiveness to different type of
abiotic stress (Robinson et
al. 1991; Bates and Lynch 2001).
Therefore, root traits are of great importance in plants for the normal growth,
yield formation and adaptive to stress conditions.
Rice is the main food for most the world’s population
and vulnerable to various abiotic stress including saline-alkaline stress (Munns and Tester 2008; Lv et al. 2013). SA stress suppress yield formation of rice
plants by inhibiting plant growth (Abbasi et al. 2015; Liu et al.
2014, 2015), damaging root system (Zhang et al. 2017) and disrupting
physiological metabolism (Liu et al. 2020). Seed germination (Lv et
al. 2013; Feng et al. 2016; Zhao et al. 2018), photosynthesis
(Xu et al. 2019), physiological metabolism (Liu et al. 2015,
2020) and yield formation (Wang et al. 2016) of rice are significantly
suppressed under salt stress conditions, and these suppression range was more
serious along with the rise of salt concentration (Lv et al. 2013).
Alkaline stress damage rice plants directly (Wei et
al. 2015) and result in deficiencies of numerous primary nutrient or
microelement, such as Fe and P (Tian et al. 2016; Liu et al.
2019). In addition, alkaline stress severely damage to rice roots by inhibiting
root growth (Lv et al. 2013), striking out
of new roots (Feng et al. 2016), damaging
root cells and reducing root vigor (Zhang et al. 2017; Liu et al.
2019). Alkaline stress results in barely new radicles striking out in the germinating
rice seeds compared to salt stress (Feng et al. 2016). The damaging
effects on rice roots by alkaline stress are associated with over accumulation
of O2•- and H2O2 induced by alkaline stress (Zhang et
al. 2017). Thus, certain differences exist in rice plants in response to
saline stress or alkaline stress, especially in root growth. However, the
mechanisms behind how rice root system response to saline or alkaline stress
remains largely unknown.
Numerous studies have demonstrated rice root growth response
to multiple managements and stress factors (Lv et al. 2013, 2014; Gu et
al. 2017; Zhang et al. 2017; Kim et al. 2020). We previously found that severe inhibition of root
growth in rice seeds (Feng et al.
2016) or seedlings (Lv et al. 2013)
were showed under NaCl stress, while more severe under alkaline stress
stimulated by Na2CO3 stress. In addition, alkaline stress
caused obvious injury of cell activity and upregulated the gene expression of
cell death pathway in rice roots (Lv et al. 2013). And root growth
indices of rice seedlings showed significant correlation to the saline-alkaline tolerance degree of different rice
varieties and represented a series of useful parameters for evaluating the
saline-alkaline stress tolerance (Lv et al. 2014). Our previous studies
showed that excess accumulation of O2•- and H2O2
induced by alkaline stress in rice roots severely damaged root cells and
decreased root activity (Zhang et al. 2017), which indicated that
oxidative stress induced by alkaline stress may be a major factor for root
damage under alkaline stress. Furthermore, SA
stress induced excess accumulation of osmolyte, such as proline and
soluble sugar, and toxic ions, such as Na+, Cl- (Liu et
al. 2020), as well as ROS (Liu et al. 2019). Changes in different
physiology traits were the results of different stress factor, such as osmolyte
was mainly induced by osmotic stress, accumulation of Na+ and Cl-
resulted in high ion toxicity (Munns and Tester 2008), and ROS accumulation
resulted in oxidative stress (Zhang et al. 2017; Liu et al.
2019). But the correlation between physiology
metabolism and root status under SA stress still
remains unknown.
This study aimed to investigate the main suppression
factor for root growth by analyzing their growth and some physiological traits
in rice seedlings under different type of SA stress conditions. This study
showed that SA stress caused remarkable inhibition to root growth as shown by
decrease of the length, surface area, diameter and volume of rice root, as well
as new root tips, and Na2CO3 stress caused more injury to
root due to its high pH. Inhibition of root growth
under SA stress is associated with osmotic stress, ion toxicity and
oxidative stress induced by saline-alkaline stress and oxidative stress was
main limiting factor which inhibited root growth in rice seedlings. This study
would provide theoretical basis for improving rice tolerance to SA stress and breeding strategy of saline-alkaline
tolerant rice varieties.
Materials and Methods
Plant materials
Four conventional japonica
rice (Oryza sativa subsp. japonica) cultivars, Dongdao-4
(D4), Changbai-9 (C9), Jinongda-19 (J19) and Nipponbare (NB) were used in
this study. The rice cultivars Dongdao-4 (D4) and Changbai-9 (C9) are
saline-alkaline tolerant rice cultivars and Jinongda-19 (J19) and Nipponbare
(NB) are sensitive to saline-alkaline stress (Liu et al. 2020).
Rice growth conditions and the stress
treatments
Rice seeds were surface-sterilized with 75% (v/v)
alcohol for 5 min, and then rinsed with deionized water five times. Full seeds
were immersed in distilled water for 2 d, and then sprinkled onto wet filter
paper in a petri dish for the germination for 24 h at 28°C in a dark incubator.
Eighteen uniformly germinated seeds were transplanted onto a multi-well plate
floating on a 320 mL cup containing deionized water for 7 d. After which, the
rice seedlings were grown in half-strength Kimura B nutrient solutions for
another 7 d (Miyake and Takahashi 1983) in a controlled growth chamber. The
growth condition was as followed: 25°C day/20°C night, and photoperiods of 12
h, 350 μmol
m-2 s-1 of photon flux intensity. Two-week-old rice
seedlings were transplanted into black buckets with
the space of 4 cm, diameter and height of bucket was 15 and 20 cm,
respectively. Rice root fixed with absorbent cotton was inserted into a
perforated black plastic foam plate. Nine uniformly rice plants were put into
buckets with different type of SA stress, which was simulated by 120 mM NaCl, 60 mM Na2SO4, 30 mM NaHCO3 and 15 mM
Na2CO3, respectively. Rice plants grown in the distilled
water were set as the control (CK), and the stress solution was replaced once
every 2 days. All rice plants were grown in a controlled growth chamber under
the growing condition with four biological replicates.
Measurement of root growth traits
Root growth traits were measured at 0, 3, 5 and 7 d of
SA stress, respectively. Rice roots were sampled for the measurement of
physiology indices after 7 d of different type of SA stress.
Rice seedlings were scanned using a root scanner of
Epson Expression 10000XL (Epson America Inc., Long Beach, CA, United States) at
0, 3, 5 and 7 d of different salt stress. The images of rice seedlings were
digitized using the WinRHizo program, according to the manufacturer’s instructions
(Regent Instruments Canada Inc., Ville de Québec, QC, Canada), and the total
root length (TRL), total root surface area (RSA), total root volume (TRV),
average root diameter (ARD) and root number (RN) were determined. The decrease
percentage of each root growth index was to evaluate the influence of different
salt stress to rice seedlings between 0 and 7 d and calculated by 100*(0–7 d)/0
d.
Measurement of Na+ and K+
content
The sodium (Na+) and potassium (K+)
contents in rice roots were measured according to Liu et al. (2020). The
dry roots samples were digested completely with the mixture of HNO3 and
HClO4 (v/v = 2:1), and then diluted to 50 mL with deionized water.
The Na+ and K+ concentrations were determined by flame
emission spectrometry (FP6410, Shanghai precision and scientific instrument
Co., Ltd., China).
Measurement of proline and soluble
sugar content
Dried rice roots of 0.1 g with 10 mL deionized water was
placed into a centrifuge tube After centrifuge, and boiling, sample
was used for the measurement of proline and soluble sugars in roots. Proline
contents were measured by the sulfosalicylic acid method, and soluble sugars
was detected with anthrone colorimetry
(Liu et al. 2020).
Measurement of ROS accumulation
Measurement of the O2•- contents was
followed by the monitoring the nitrite formation from hydroxylamine in the
presence of O2•-, described by Elstner and Heupel (1976) and
Jiang and Zhang (2001). The H2O2 contents were measured
by monitoring content of the titanium-peroxide complex at A415 according
to the Brennan and Frenkel (1977), Zhang et al. (2017) and Liu et al.
(2019).
Experimental design and data analyses
The statistical software SPSS 21.0 (IBM Corp., Armonk,
NY) was used in the statistical analyses. Based on the results of one-way
analysis of variance (ANOVA), significant difference (P < 0.05) was compared
among different rice varieties or treatments using Duncan’s multiple range test
(DMRT).
Results
Root growth
traits
Total root length: Root elongation
of rice seedlings were significantly inhibited as shown by the decrease of
total root length under different type of salt stress conditions (Fig 1). Total
root length of the saline-alkaline tolerant cultivars, D4 and
C9, were higher than the sensitive cultivars, J19 and
NB (Fig 1b–e), during the stress. The average descend percentage compared to 0 d
under NaCl, Na2SO4, NaHCO3 and Na2CO3
conditions were 28.4, 22.6, 21.8 and 32.6%, respectively (Fig. 1f). Na2CO3 stress caused
the most serious inhibition to root elongation of rice seedlings as shown by
the maximal fold change under Na2CO3 stress (Fig. 1f).
Fig. 1: Total root length of four rice varieties (D4, C9, J19, NB) were measured
at 0 d, 3 d, 5 d, and 7 d, under CK (a),
NaCl (b), Na2SO4 (c), NaHCO3 (d), and Na2CO3 (e) conditions. Descend percentage of root
length of 7 d (f) was expressed by taking
values of 0 d as 1 under different stress conditions (0d-7d/0d). Values are
means ± SD, n=4. Different letters on
the column represent significant difference (P < 0.05) between different rice varieties based on Duncan’s
test
Fig. 2: Total root surface area of four
rice varieties (D4, C9, J19, NB) were measured at 0 d, 3 d, 5 d, and 7 d, under
CK (a), NaCl (b), Na2SO4 (c), NaHCO3
(d), and Na2CO3 (e) conditions. Descend percentage of root surface
area of 7 d (f) was expressed by taking values of 0 d as 1 under
different stress conditions (0d-7d/0d). Values are means ± SD, n=4. Different letters on the column
represent significant difference (P<0.05)
between different rice varieties based on Duncan’s test
Fig. 3: Total root volume of four rice varieties (D4, C9, J19,
NB) were measured at 0 d, 3 d, 5 d, and 7 d, under CK (a), NaCl (b),
Na2SO4 (c), NaHCO3 (d), and Na2CO3
(e) conditions. Descend
percentage of root volume of 7 d (f) was expressed by taking
values of 0 d as 1 under different stress conditions (0d-7d/0d). Values are
means ± SD, n=4. Different letters on
the column represent significant difference (P < 0.05) between different rice varieties based on Duncan’s
test
Total root surface area
Change of total root surface area was similar to root
length under different type salt stress factors as shown by salt stress caused
a significant downward trend of root surface area of the four rice cultivars
(Fig. 2). Total root surface area in the saline-alkaline sensitive cultivars,
J19 and NB, were lower, and the descend range were higher than the
saline-alkaline tolerant cultivars, D4 and C9, respectively (Fig. 2b–e). The average descend range of root surface area in four
cultivars was 26.2, 24.2, 20.6 and 29.3%, under NaCl, Na2SO4,
NaHCO3 and Na2CO3 conditions, respectively (Fig. 2f).
Total root volume
A remarkable decrease of root volume was observed under SA stress conditions compared to CK, and root volume of the saline-alkaline
tolerant cultivars were higher than the sensitive cultivars (Fig. 3). Average decrease range of the
root volume in four cultivars were 38.7, 37.9, 37.2, and 40.9% under NaCl, Na2SO4, NaHCO3
and Na2CO3 conditions, respectively (Fig. 4f), and it was higher than root length
and surface area, which indicated that more serious damage was showed in root
volume under stress conditions.
Average root diameter
Saline or alkaline stress caused remarkable decrease of
root diameter in the four rice cultivars (Fig 4a–e). The
saline-alkaline tolerant cultivars showed higher root diameter than the
sensitive cultivars. But the average descend range of the root diameter in four cultivars was 29.7% under NaCl condition, which was higher than other
stress
factors, while it was 19.0, 21.1 and 21.4% under Na2SO4, NaHCO3 and Na2CO3
conditions, respectively (Fig 4f).
These results indicated that NaCl stress caused more damage on root diameter.
Root numbers
Fig. 4: Average root diameter of four rice varieties (D4, C9,
J19, NB) were measured at 0 d, 3 d, 5 d, and 7 d, under CK (a), NaCl (b),
Na2SO4 (c), NaHCO3 (d), and Na2CO3
(e) conditions. Descend percentage
of root diameter of 7 d (f) was expressed by taking values of 0 d
as 1 under different stress conditions (0d-7d/0d). Values are means ± SD, n=4. Different letters on the column
represent significant difference (P < 0.05)
between different rice varieties based on Duncan’s test
Saline or alkaline stress caused serious damage on root
numbers compared to CK (Fig. 5a–e), especially under Na2CO3 condition, in which the average descend range was 49.6% (Fig. 5f). Average descend range of root
numbers was 41.3, 37.2 and 36.1% under NaCl, Na2SO4, and NaHCO3 conditions, respectively (Fig. 5f). Numbers of new roots were lower
in the saline-alkaline sensitive cultivars compared with the tolerant
cultivars, and the descend range of sensitive cultivars were higher as well
(Fig. 5a–e).
Root physiological traits
Accumulation
of osmolytes, ions and ROS in rice roots: Saline or alkaline
stress induced excessive accumulation of Na+ in roots, while K+
contents significantly decreased under stress conditions compared to CK (Fig 6a–b). Rice roots showed a higher Na+ and lowed K+
accumulation under NaCl and Na2CO3 treatments, indicating
that severe ion toxicity occurred to roots by NaCl and Na2CO3
treatment. The total Na+ content in the saline-alkaline tolerant
varieties (D4 and C9) was deceased by 11.3, 9.1, 4.8 and 8.5% compared to the
sensitive varieties (J19 and NB) at NaCl, Na2SO4, NaHCO3
and Na2CO3 treatment (Fig 6a), respectively. And the K+ content increased by 4.6,
5.8, 9.8 and 1.8% at NaCl, Na2SO4, NaHCO3 and
Na2CO3 treatment (Fig 6b), respectively.
The osmolytes, such as proline and soluble sugar,
accumulated under different salt stress conditions, and the accumulation of these osmolytes in saline-alkaline sensitive cultivars,
J19 and NB, was higher than tolerant cultivars, D4 and C9 (Fig. 6c–d). The increase of these osmolytes in the NaCl treatment was the
highest of all the stress factors compared to CK (Fig. 6c–d).
Consistently, over accumulation of O2•- and H2O2 was observed in rice roots, and more accumulation in the
saline-alkaline sensitive cultivars J19 and NB (Fig 6e–f). The O2•- content in Na2CO3 and NaHCO3 treatments increased by 29.5‒38.4% compared to the NaCl or Na2SO4 treatments, and H2O2 content by 16.4‒26.2%, respectively
(Fig. 6e–f). These results indicated that alkaline stress caused more ROS
in rice roots than no salt stress.
Correlation between the growth indices and
physiological traits in roots
Correlation analysis showed that the growth indices of
roots and physiological traits were statistically significant. The TRL,
Fig. 5: Root numbers of four rice varieties (D4, C9, J19, NB)
were measured at 0 d, 3 d, 5 d, and 7 d, under CK (a), NaCl (b),
Na2SO4 (c), NaHCO3 (d), and Na2CO3
(e) conditions. Descend
percentage of root numbers of 7 d (f) was expressed by taking
values of 0 d as 1 under different stress conditions (0d-7d/0d). Values are
means ± SD, n=4. Different letters on
the column represent significant difference (P < 0.05) between different rice varieties based on Duncan’s
test
Fig. 6: Content of root Na+ (a), K+
(b), proline (c), soluble sugar (d), O2`-
(e) and H2O2 (f) of four rice varieties
(D4, C9, J19, NB) were measured at 7 d under CK, NaCl, Na2SO4,
NaHCO3, and Na2CO3 treatments. Values are means
± SD, n=4. Different letters on the
column represent significant difference (P
< 0.05) between different rice varieties based on Duncan’s test
RSA, and ARD of the NaCl treatment showed significant
negative correlation to Na+, proline, soluble sugar, O2•- and H2O2 content, except for K+
content (Table 1a). The TRV showed
significant negative correlation to Na+,
proline, O2•- and H2O2
content, except for soluble sugar and K+
content (Table 1a). The RN showed significant negative correlation to proline,
soluble sugar, O2•- and H2O2 content, except for Na+ and
K+ content (Table 1a).
The TRL, RSA, and TRV of the Na2SO4
treatment showed significant negative correlation to proline, O2•- and H2O2 content, except for Na+
soluble sugar and K+ content (Table 1b). The ARD showed significant negative correlation to Na+,
proline, O2•- and H2O2
content, except for soluble sugar and K+ content
(Table 1b). The RN showed
extremely significant or significant negative correlation to soluble sugar, O2•- and H2O2 content, except for Na+,
proline, and K+ content (Table 1b).
The TRL, RSA and ARD of the NaHCO3 treatment
showed significant negative correlation to proline, soluble sugar, O2•- and H2O2 content, except for Na+
content (Table 1c). The TRL and
RSA showed significant positive correlation to K+ content (Table 1c). The TRV showed significant
negative correlation to proline, O2•- and H2O2
content, except for Na+, K+ and soluble sugar content
(Table 1c). The RN showed
significant negative correlation to soluble sugar, O2•- and H2O2 content, except for Na+, K+
and proline content (Table 1c).
The TRL and TRV of the Na2CO3
treatment showed significant negative correlation to proline, O2•- and H2O2 content, except for Na+, K+
and soluble sugar content (Table 1d).
The RSA, ARD and RN showed significant negative correlation to Na+,
proline, O2•- and H2O2 content, except for K+ and
soluble sugar content (Table 1d).
Discussion
SA stress is a complex stress factor, including high
salinity, osmotic pressure and pH inhibiting plants growth and yield formation
by multiple ways (Wei et al. 2015; Liu et al. 2016; Wang et al.
2018). Root plays the key role in the uptake of water and nutrients from soil
in plants. SA stress changed root morphology and architecture (Liu et al.
2016), as well as root growth as shown by decreasing root length, volume, new
root tips and surface area of many crops (Neves et al. 2010; Lv et al.
2014; Guo et al. 2016; Zhang et al. 2017), which may be
associated to root lignin levels induced by stress (Lin and Kao 2001). In
addition, SA stress disturbed root metabolism system by damaging cell activity
(Zhang et al. 2017), upregulating
transcription expression of cell death indication genes (Lv et al. 2013), resulting in
overaccumulation of Na+ and
ROS (Guo et al. 2016; Wei et
al. 2015). In this study,
root growth of rice seedlings was significantly inhibited by multiple SA stress
(Fig. 1‒5), and SA stress significantly induced excess accumulation of Na+,
proline, soluble sugar, and ROS (Fig. 6). In addition, inhibition of root
growth was closely associated with high osmotic pressure, ion toxicity and
oxidative stress as shown by overaccumulation of Na+, proline,
soluble sugar, H2O2 and O2•- (Table 1).
These data collectively suggest that root growth condition is vital for plants
responses to SA stress.
Table 1: Correlation analysis of root growth and physiological
traits under stress conditions
Stress |
TRL |
RSA |
TRV |
ARD |
RN |
Na+ |
K+ |
PC |
SSC |
O2•- |
|
(a) NaCl |
RSA |
0.886** |
|||||||||
TRV |
0.914** |
0.899** |
|||||||||
ARD |
0.753** |
0.798** |
0.657** |
||||||||
RN |
0.469 |
0.596* |
0.618* |
0.414 |
|||||||
Na+ |
-0.732** |
-0.663** |
-0.728** |
-0.530* |
-0.319 |
||||||
K+ |
0.300 |
0.141 |
0.202 |
0.440 |
0.202 |
0.110 |
|||||
PC |
-0.736** |
-0.805** |
-0.811** |
-0.677** |
-0.646** |
0.336 |
-0.349 |
||||
SSC |
-0.514* |
-0.607* |
-0.438 |
-0.682** |
-0.689** |
0.186 |
-0.283 |
0.635** |
|||
O2•- |
-0.788** |
-0.870** |
-0.852** |
-0.673** |
-0.809** |
0.537* |
-0.313 |
0.747** |
0.633** |
||
H2O2 |
-0.854** |
-0.904** |
-0.941** |
-0.720** |
-0.696** |
0.742** |
-0.141 |
0.767** |
0.520* |
0.840** |
|
(b) Na2SO4 |
RSA |
0.851** |
|||||||||
TRV |
0.791** |
0.860** |
|||||||||
ARD |
0.780** |
0.842** |
0.843** |
||||||||
RN |
0.501* |
0.577* |
0.427 |
0.485 |
|||||||
Na+ |
-0.492 |
-0.398 |
-0.493 |
-0.569* |
-0.187 |
||||||
K+ |
0.122 |
0.145 |
0.314 |
0.195 |
0.174 |
-0.036 |
|||||
PC |
-0.742** |
-0.643** |
-0.693** |
-0.562* |
-0.041 |
0.493 |
-0.254 |
||||
SSC |
-0.240 |
-0.352 |
-0.311 |
-0.388 |
-0.749** |
0.370 |
-0.264 |
-0.0110 |
|||
O2•- |
-0.724** |
-0.795** |
-0.746** |
-0.747** |
-0.507* |
0.657** |
-0.024 |
0.724** |
0.424 |
||
H2O2 |
-0.807** |
-0.792** |
-0.815** |
-0.753** |
-0.600* |
0.555* |
-0.182 |
0.706** |
0.494 |
0.864** |
|
(c) NaHCO3 |
RSA |
0.947** |
|||||||||
TRV |
0.832** |
0.911** |
|||||||||
ARD |
0.815** |
0.823** |
0.783** |
||||||||
RN |
0.392 |
0.476 |
0.329 |
0.522* |
|||||||
Na+ |
-0.390 |
-0.376 |
-0.368 |
-0.295 |
-0.479 |
||||||
K+ |
0.646** |
0.542* |
0.440 |
0.309 |
0.217 |
-0.124 |
|||||
PC |
-0.661** |
-0.676** |
-0.622* |
-0.517* |
-0.136 |
0.479 |
-0.322 |
||||
SSC |
-0.603* |
-0.557* |
-0.461 |
-0.631** |
-0.697** |
-0.200 |
-0.457 |
0.240 |
|||
O2•- |
-0.739** |
-0.784** |
-0.771** |
-0.760** |
-0.545* |
0.139 |
-0.493 |
0.646** |
0.673** |
||
H2O2 |
-0.893** |
-0.926** |
-0.802** |
-0.861** |
-0.668** |
0.187 |
-0.500* |
0.701** |
0.664** |
0.847** |
|
(d) Na2CO3 |
RSA |
0.879** |
|||||||||
TRV |
0.866** |
0.868** |
|||||||||
ARD |
0.762** |
0.902** |
0.780** |
||||||||
RN |
0.868** |
0.853** |
0.801** |
0.684** |
|||||||
Na+ |
-0.446 |
-0.515* |
-0.464 |
-0.597* |
-0.508* |
||||||
K+ |
0.001 |
0.275 |
0.159 |
0.278 |
0.116 |
-0.474 |
|||||
PC |
-0.739** |
-0.839** |
-0.767** |
-0.826** |
-0.715** |
0.692** |
-0.308 |
||||
SSC |
-0.044 |
-0.263 |
-0.150 |
-0.278 |
-0.036 |
-0.128 |
0.106 |
0.384 |
|||
O2•- |
-0.608* |
-0.706** |
-0.552* |
-0.672** |
-0.572* |
0.168 |
0.012 |
0.774** |
0.614* |
||
H2O2 |
-0.651** |
-0.728** |
-0.754** |
-0.599* |
-0.536* |
0.184 |
0.077 |
0.688** |
0.611* |
0.689** |
†TRL:
Total root length, RSA: Total root surface area, TRV: Total root volume, ARD:
Average root diameter, RN: Root numbers, Na+: Na+ content,
K+: K+ content, PC: Proline content, SSC: Soluble sugar
content, O2·-: O2·- content, H2O2:
H2O2 content
†The correlation coefficient (r2) was showed in the
table; ** indicates significant difference at P < 0.01 level;
* indicates significant difference at P < 0.05 level
Numerous studies have showed that root growth and
physiological metabolism under various stresses conditions (Redjala et al.
2011; Lv et al. 2013; Shi et al. 2015; Liu et
al. 2016; Kim et al. 2020). Rice root growth, architecture and
root-to-shoot relationship was changed by water deficit (Pérez-Alfocea et al.
2011; Kim et al. 2020), and osmoregulatory substances played vital role
in the regulation of osmotic pressure in roots (Sharma and Dietz 2006). Under
SA stress, differences were showed in the response to neutral salts and carbonates of roots. Seed germination of rice was
remarkably inhibited by salt and alkali stress, as well as the growth of shoots
or roots, but barely new root tips were sprouted in the germinated seeds (Feng et
al. 2016). In addition, alkali stress caused severer cell injury of rice
seedlings as shown by the more significant expression of cell death-related
genes induced by alkali stress (Lv et al. 2013; Zhang et al.
2017). A significant inhibition in rice root growth was showed in all the SA
stress factors by severe decrease of TRL (Fig. 1), RSA (Fig. 2), TRV (Fig. 3)
and ARD (Fig. 4), RN (Fig. 5) in this study. The TRL, RSA, TRV and RN was
inhibited severer by Na2CO3 treatment (Fig 1‒3, 5), but ARD was
influenced more seriously by NaCl treatment (Fig. 4). These data suggested
alkali stress caused more serious injury to root system of rice seedlings than
salt stress, which was possible due to the high pH induced by carbonates,
resulting in disorder or deficiencies of nutritional minerals around root (Tian
et al. 2016; Liu et al. 2019). Furthermore, fewer new root tips
observed under alkali stress, which was mainly due to more serious of cell
death induced by alkali stress (Zhang et al. 2017).
Previous studies have investigated the physiological
reaction of plants response to stress, and plants adapt to various
environmental stress factors by regulating multiple physiological metabolic
processes, such as ion transport, osmoregulation,
ROS-scavenging and gene transcription (Kim et al. 2020; Liu et al.
2020). SA stress caused excessive accumulation of toxic ions, such as Na+
and Cl-, which resulted in the damaging of leaf photosynthetic
structure and decline in photosynthetic efficiency in rice (Liu et al.
2021). While increase of K+ content contributed to block the Na+
entrance path into cell and high K+/Na+ rate was
observed in the salt tolerant rice varieties (Peng et al. 2004; Lv et
al. 2013). In this study, SA stress resulted in a significant increase of
Na+ and decreased K+ in roots, and more Na+
content was observed in NaCl and Na2CO3
treatments (Fig. 6a‒6b), indicating that SA stress caused
disbalance of ion homeostasis in cells and overaccumulation of Na+
induced high ion toxicity to rice roots. Osmoregulation is an important
regulation mechanism and physiological response of plants to various stress
conditions (Lv et al. 2014; Liu et al. 2015). Many plants
accumulate osmotica, such as proline and soluble sugar, and proline content has
been used as a selection parameter to evaluate the stress tolerance of plants
(Székely et al. 2008). However, in rice, proline content and the
fold-change of proline accumulation showed no significant correlation to
tolerance of rice varieties under different SA stress factors, indicating that
proline accumulation was a result of SA stress (Lv et al. 2014). In the
present study, high osmotic pressure was induced by SA stress as evident by a
remarkable increase of proline and soluble sugar in roots (Fig. 6c‒6d), and the most content of proline and soluble was observed in
the NaCl treatment. In addition, proline and soluble sugar contents in the
sensitive varieties (J19 and NB) were higher than the tolerant varieties (D4
and C9), supposing that the osmotic adjustment system was affected by SA
stress. Previous studies showed that overaccumulation of ROS is an important
injury factor to rice under alkaline stress and alkaline tolerance in rice was
associated to ROS-scavenging capability (Guo et al. 2014; Guan et al.
2017; Zhang et al. 2017). Results of this study showed that SA stress
caused overaccumulation of ROS in roots of rice seedlings, such as O2•- and H2O2 (Fig 6e‒f), and the most accumulation was in Na2CO3
treatment, indicating that severe oxidative stress was induced by alkali stress
compared to salt stress.
Plants grown in saline-alkaline soil suffer from a complex abiotic
stress, stimulated by single or multiple combinations of sodium
salt, such as NaCl, Na2SO4, NaHCO3 and Na2CO3.
Previous studies have showed that response of plants to different sodium stress
is a complex network (Ahmed et al. 2020; Wang and Jiang 2020). Our
results showed that root growth indices were significant correlated to
accumulation of ion, osmolytes and ROS in roots (Table 1). The ROS accumulation
in root was significant negative correlated to root growth indices under all SA
stress factors indicating oxidative stress was induced by different sodium salt
stress, which was the same limitation factor damaging rice roots under SA
stress conditions. In addition, there were differences under single sodium salt
treatment except for ROS accumulation. Under NaCl treatment, root growth was
inhibited generally by ion toxicity and osmotic stress as shown by good
correlations with Na+, proline and soluble sugar contents (Table 1a). Under Na2SO4
treatment, proline content showed good correlation to root growth indices
(Table 1b). But root growth had
significant correlation to K+ accumulation and osmotic adjustment
under NaHCO3 treatment, which suggested that
osmotic stress also suppressed root growth (Table 1c). While under Na2CO3 stress, proline and
Na+ content showed significant correlation to most root growth
indices, indicating osmotic stress and ion toxicity caused injury to root
system, especially in RSA, ARD and RN (Table 1d). These results suggested that excess accumulation of Na+
and ROS in root accounted for lower resistance to SA stress in rice seedlings.
Overaccumulation of ROS in rice roots caused severe damage to cell membrane at
seed germination (Zhao et al. 2021) and seedling stage (Zhang et al.
2017), which directly inhibited seed germination and seedlings growth; while
massive accumulation of Na+ caused severe
damage to the leaf photosynthetic structure in rice (Liu et al. 2021).
Hence, decrease of ROS accumulation and increase of K+/Na+
rate under SA stress conditions by multiple methods will be a potential
approach to improve tolerance to stress factor and a focused point for the
breeding strategy in the future.
Rice varieties at different degree of saline-alkaline tolerance vary in
different growth and physiological traits. Previous studies showed that rice varieties with
higher tolerance to SA stress exhibited better growth status and physiological
metabolism as shown by higher survival rate, good root growth indices and lower
ROS or Na+ content (Lv et al. 2014; Feng et al. 2016;
Zhang et al. 2017). However, proline content was insufficient to serve
as reliable physiological traits to evaluate the tolerance to SA stress among
rice varieties (Lv et al. 2014). In the present study, we selected two saline-alkaline tolerant rice cultivars, D4 and C9, and
two saline-alkaline sensitive rice cultivars, J19 and NB (Feng et al.
2016; Liu et al. 2020). These four rice cultivars exhibited different
changes in root growth indices in response to SA
stress as evident in higher TRL, RSA, TRV, ARD and RN and lower decrease
observed in D4 and C9 under different types of SA stress (Fig
1‒5). These results
suggest that the saline-alkaline tolerant rice cultivars
are based on the differences in root growth indices, as reported earlier (Lv et
al. 2013, 2014). Significantly lower accumulation of Na+, O2•- and H2O2, as well as higher K+ (Fig.
6), in the rice cultivars D4 and C9, improved tolerance to SA stress by
decreasing ion toxicity and oxidative stress (Peng et al. 2004; Kanawapee et al. 2012; Zhang et al. 2017;
Liu et al. 2019). However, accumulation of proline and soluble sugar in the saline-alkaline
sensitive rice cultivars was higher than in the tolerant varieties (Fig. 6c‒6d), which may indicate that proline accumulation is a symptom
and referent of poor osmotic adjustment capability in the saline-alkaline
sensitive rice cultivars (Vaidyanathan et al. 2003; Kanawapee et al.
2012).
Conclusion
In summary, SA stress caused severe inhibition in root
growth of rice seedlings, resulting in overaccumulation of Na+,
proline, soluble sugar and ROS in rice roots, indicated that high ion toxicity,
osmotic stress and oxidative stress to rice roots was induced by SA stress.
Furthermore, root growth inhibition under SA stress conditions was associated
to osmotic stress, ion toxicity and oxidative stress
induced by SA stress. In addition, oxidative stress induced by saline-alkaline
stress was the main restrictive factor that inhibited root growth in rice
seedlings.
Acknowledgements
This work is supported by the Science and Technology
Project of Education Department of Jiangxi Province (Project Numbers:
GJJ190868); the Natural Science Foundation of Jiangxi Province (Project
Numbers: 20202BABL213046); the Science and Technology Development Projects of
Jilin Province (Project Numbers: 20200702013NC and 20200402027NC) for their
financial support.
Author Contributions
X-LL, CX, and H-JW designed the study; X-LL, H-TY, P-P
S, QS, NC and L-NL performed the laboratory experiments and measurement of the
indices; X-LL and CX performed the data collection, analysis and figure
mapping; X-LL and CX wrote the manuscript; H-JW participated in the
modification of the manuscript; X-LL, Z-AZ and H-JW provided scientific
expertise.
Conflict of Interest
The authors declare that they have no
competing financial interests.
Data Availability
The data used to support the findings
of this study are available from the corresponding author upon request.
Ethics Approval
Not applicable to this paper
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