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 HNO₃and 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 H₂O₂contents were measured by monitoring content of the titanium-peroxide complex at A₄₁₅according 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), O_2`^- (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	Index	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

References

Abbasi GH, A Javaid, A Rafiq, J Moazzam, A Muhammad, A Shafaqat, I Muhammad (2015). Potassium application mitigates salt stress differentially at different growth stages in tolerant and sensitive maize hybrids. Plant Growth Regul 76:111–125

Ahmed S, YH Shen, WH Ma, JB Dong, XY Qu, Y Cao, SL Hu, XG Luo (2020). Effects of three-salt stress on the growth of maize seedlings and the accumulation of antioxidant enzymes and ions in leaves. Agric Res Arid Area 38:112–117

Bates TR, JP Lynch (2001). Root hairs confer a competitive advantage under low phosphorus availability. Plant Soil 236:243–250

Brennan T, C Frenkel (1977). Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiol 59:411–416

Comas LH, KE Mueller, LL Taylor, PE Midford, HS Callahan, DJ Beerling (2012). Evolutionary patterns and biogeochemical significance of angiosperm root traits. Intl J Plant Sci 173:584–595

Elstner EF, A Heupel (1976). Inhibition of nitrite formation from hydroxylammonium chloride: A simple assay for superoxide dismutase. Anal Biochem 70:616–620

FAO (2016) Management of Salt Affected Soils. Food and Agriculture Organization of the United Nations. http://www.fao.org/soils-portal/soil-management/management-of-someproblem-soils/salt-affected-soils/more-information-on-saltaffected-soils/en/

Feng ZH, XL Liu, CJ Jiang, ZW Liang (2016). Comprehensive evaluation of rice (Oryza sativa japonica) germplasm for saline/alkaline tolerance at germination stage from Jilin province, China. Soils Crops 5:120‒127

Ghosh D, J Xu (2014). Abiotic stress responses in plant roots: A proteomics perspective. Front Plant Sci 5; Article 6

Gu D, F Zhen, DB Hannaway, Y Zhu, L Liu, W Cao, L Tang (2017). Quantitative classification of rice (Oryza sativa L.) root length and diameter using image analysis. PLoS One 12; Article e0169968

Guan Q, X Liao, M He, X Li, Z Wang, H Ma, S Yu, S Liu (2017). Tolerance analysis of chloroplast OsCu/Zn-SOD overexpressing rice under NaCl and NaHCO₃ stress. PLoS One 12; Article e0186052

Guo M, R Wang, J Wang, K Hua, Y Wang, X Liu, S Yao (2014). ALT1, a Snf2 family chromatin remodeling ATPase, negatively regulates alkaline tolerance through enhanced defense against oxidative stress in rice. PLoS One 9; Article e112515

Guo P, H Wei, W Zhang, Y Bao (2016). Physiological Responses of Alfalfa to high-level salt stress: Root ion flux and stomatal characteristics. Intl J Agric Biol 18:125‒133

Hernández E, A Vilagrosa, J Pausas, J Bellot (2010). Morphological traits and water use strategies in seedlings of mediterranean coexisting species. Plant Ecol 207:233–244

Jiang MY, JH Zhang (2001). Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings. Plant Cell Physiol 42:1265–1273

Kaashyap M, R Ford, H Kudapa, M Jain, D Edwards, R Varshney, N Mantri (2018). Differential regulation of genes involved in root morphogenesis and cell wall modification is associated with salinity tolerance in chickpea. Sci Rep 8; Article 4855

Kanawapee N, J Sanitchon, W Lontom, P Threerakulpisut (2012). Evaluation of salt tolerance at the seedling stage in rice genotypes by growth performance, ion accumulation, proline and chlorophyll content. Plant Soil 358:235–239

Kim Y, YS Chung, E Lee, P Tripathi, S Heo (2020). Root response to drought stress in rice (Oryza sativa L.). Intl J Mol Sci 21:1513–1534

Koevoets IT, JH Venema, JT Elzenga, C Testerink (2016). Roots withstanding their environment: Exploiting root system architecture responses to abiotic stress to improve crop tolerance. Front Plant Sci 7; Article 1335

Lin, CC, H Kao (2001). Cell wall peroxidase against ferulic acid, lignin, and NaCl-reduced root growth of rice seedlings. J Plant Physiol 158:667–671

Liu M, ZW Liang, LH Huang, MM Wang, HY Yang (2016). An effective method for estimation rice (Oryza sativa L.) crown root numbers at the heading stage in saline-sodic soils of Northeast China. Phyton 85:162–168

Liu XL, C Xu, P Ji, Q Li, HT Yang, ZH Wu, HJ Wang (2021). Correlation analysis of chlorophyll fluorescence characteristics of leaf and ions accumulation in rice under salt stress. Mol Plant Breed 19:972–982

Liu XL, C Xu, P Ji, Q Li, M Zhu, Z Zhang, F Ling, H Wang (2020). Lower nitrogen levels improve growth and some physiological traits of rice (Oryza sativa) under salt stress during reproductive period. Intl J Agric Biol 24:769‒776

Liu XL, H Zhang, YY Jin, MM Wang, HY Yang, HY Ma, CJ Jiang, ZW Liang (2019). Abscisic acid primes rice seedlings for enhanced tolerance to alkaline stress by upregulating antioxidant defense and stress tolerance-related genes. Plant Soil 438:39‒55

Liu XL, C Xu, KZ Xu, JJ Cui, ZA Zhang, FL Ling, JH An, ZH Wu (2015). Effects of salt stress on photosynthetic characteristics and some physiological traits of rice varieties at different nitrogen levels. J South Chin Agric Univ 36:6‒12

Liu XL, C Xu, KZ Xu, JJ Cui, JH An, FL Ling, ZA Zhang, ZH Wu (2014). Effects on characteristics of photosynthesis and chlorophyll fluorescence of rice under salt stress. Crops 2:88‒92

Lv BS, HY Ma, XW Li, LX Wei, HY Lv, HY Yang, CJ Jiang, ZW Liang (2014). Proline accumulation is not correlated with saline-alkaline stress tolerance in rice seedlings. Agron J 107:51-60

Lv BS, XW Li, HY Ma, Y Sun, LX Wei, CJ Jiang, ZW Liang (2013). Differences in growth and physiology of rice in response to different saline-alkaline stress factors. Agron J 105:1119–1128

Miyake Y, E Takahashi (1983). Effect of silicon on the growth of solution-cultured cucumber plant. Soil Sci Plant Nutr 29:71–83

Munns R, M Tester (2008). Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681

Neves GYS, R Marchiosi, MLL Ferrarese, RC Siqueira-Soares, O Ferrarese-Filho (2010). Root growth inhibition and lignification induced by salt stress in soybean. J Agron Crop Sci 196:467–473

Peng YL, ZW Gao, Y Gao, GF Liu, LX Sheng, DL Wang (2008). Eco-physiological characteristics of alfalfa seedlings in response to various mixed salt-alkaline stresses. J Integr Plant Biol 50:29–39

Pérez-Alfocea F, ME Ghanem, A Gómez-Cadenas, LC Dodd (2011). Omics of root-to-shoot signaling under salt stress and water deficit. J Intl Biol 15:893‒901

Redjala T, I Zelko, T Sterckeman, V Legué, A Lux (2011). Relationship between root structure and root cadmium uptake in maize. Environ Exp Bot 71:241–248

Robinson D, D Linehan, S Caul (1991). What limits nitrate uptake from soil? Plant Cell Environ 14:77–85

Sharma SS, KJ Dietz (2006). The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress. J Exp Bot 57:711–726

Shi JC, S Li, Q Zuo, A Ben-Gal (2015). An index for plant water deficit based on root-weighted soil water content. J Hydrol 522:285–294

Székely G, E Ábrahám, A Cséplő, G Rigó, L Zsigmond, J Csiszár, F Ayaydin, N Strizhov, J, Jásik, E Schmelzer, C Koncz, L Szabados (2008). Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis. Plant J 53:11–28

Tian Z, J Li, X Jia, F Yang, Z Wang (2016). Assimilation and translocation of dry matter and phosphorus in rice genotypes affected by salt-alkaline stress. Sustain 8; Article 568

Vaidyanathan H, P Sivakumar, R Chakrabarty, G Thomas (2003). Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.)-Differential response in salt-tolerant and sensitive varieties. Plant Sci 165:1411–1418

Wang MM, P Rengasamy, ZC Wang, F Yang, HY Ma, LH Huang, M Liu, HY Yang, JP Li, FH An, YY Li, XL Liu, ZW Liang (2018). Identification of the most limiting factor for rice yield using soil data collected before planting and during the reproductive stage. Land Degrad Dev 29:2310–2320

Wang MM, F Yang, HY Ma, LX Wei, LH Huang, M Liu, HY Yang, JP Li, XW Li, XL Liu, CJ Jiang, ZW Liang (2016). Cooperative effects of sand application and flushing during the sensitive stages of rice on its yield in a hard saline-sodic soil. Plant Prod Sci 19:468‒478

Wang WH, L Jiang (2020). Effect of sodium stress on seed germination and seedling growth of Atriplex aucheri. Chin J Grassl 42:23–29

Wasson AP, RA Richards, R Chatrath, SC Misra, SV Prasad, GJ Rebetzke, JA Kirkegaard, J Christopher, M Watt (2012). Traits and selection strategies to improve root systems and water uptake in water-limited wheat crops. J Exp Bot 63:3485–3498

Wei LX, BS Lv, MM Wang, HY Ma, HY Yang, XL Liu, CJ Jiang, ZW Liang (2015). Priming effect of abscisic acid on alkaline stress tolerance in rice (Oryza sativa L.) seedlings. Plant Physiol Biochem 90:50–57

Xu C, Q Li, X Liu, HJ Wang, X Liang, F Ling, Z Wu, Z Zhang, Z Chen (2019). Effects of nitrogen supply level on photosynthesis and chlorophyll fluorescence characteristics of rice under salt stress. Emir J Food Agric 31:741–751

Yang CW, JN Chong, CY Li, CM Kim, DC Shi, DL Wang (2007). Osmotic adjustment and ion balance traits of an alkali resistant halophyte Kochia sieversiana during adaptation to salt and alkali conditions. Plant Soil 294:263–276

Zhang H, XL Liu, RX Zhang, HY Yuan, MM Wang, HY Yang, HY Ma, D Liu, CJ Jiang, ZW Liang (2017). Root damage under alkaline stress is associated with reactive oxygen species accumulation in rice (Oryza sativa L.). Front Plant Sci 8; Article 1580

Zhao H, FF Xu, AQ Xiong, F Wu, HL Zhang (2021). Effects of different salt stress on seed germination and seedling of rice. Mol Plant Breed 2021:1–7

Zhao HJ, ML Ma, FY Dai, HC Ren (2018). Effects of salt stress on germination indexes and isozymes of different rice varieties in Ningxia. Jiangsu Agric Sci 46:44–47

Zobel RW, Y Waisel (2010). A plant root system architectural taxonomy: A framework for root nomenclature. Plant Biosyst 144:507–512