Physiological evaluation for salt tolerance in green and purple leaf color rice cultivars at seedling stage

Abstract

Anthocyanin, a water-soluble pigment found in plants, has been reported to be associated with abiotic stress tolerance including salt stress. For a better understanding of the role of anthocyanin in response to salt stress, two salt-tolerant rice genotypes having different leaf anthocyanin content, one having green (‘Pokkali’; PK) and the other purple leaves (‘Niew Dam 019’; ND 019), were used in this study. After being subjected to salt stress (150 mM NaCl) for 5 d, the 3-week-old rice genotypes PK and ND 019 exhibited significant physiological responses (water content, Na⁺/K⁺ ratio, osmolyte accumulation, osmotic adjustment, antioxidant capacity, membrane damage and chlorophyll) and expression of ion transporter genes, indicating overall salt tolerance ability. However, the green-leaved rice variety, PK, had better root-to-shoot Na⁺ exclusion mechanism than the purple-leaved variety, ND 019 as evidenced by lower Na⁺ accumulation in leaves compared to ND 019 despite the fact that they accumulated the similar level of Na⁺ in roots. On the other hand, ND 019 accumulated higher concentration of osmolytes leading to more enhanced osmotic adjustment. These results revealed that Na⁺ ion exclusion was the prominent salt tolerance mechanism in the green-leaved PK whereas in the purple-leaved ND 019 osmotic adjustment was the more significant strategy. Under salt stress, there was no remarkable change in anthocyanin in PK while a reduction was found in ND 019. Thus, it could be proposed that anthocyanin did not play a vital role in protecting the purple-leaved rice, ND 019 from salt stress during seedling stage.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-021-01114-y.

Introduction

Deteriorating environmental factors following global warming are adversely affecting plant growth and crop production worldwide. Soil salinity is major abiotic stress restricting crop cultivation area in the north-eastern Thailand where the saline land exists naturally (Pongwichian 2016). Saline soil water primarily cause osmotic stress which inhibits plant growth by interfering with plant water uptake. Subsequently, ion toxicity occurs after plant dramatically accumulates sodium (Na⁺) and chloride (Cl⁻) ions leading to ionic imbalance and a reduction in cytosolic potassium ion (K⁺) uptake causing erratic metabolic processes, and nutrient deficiency which consequently affects plant growth and development (Assaha et al. 2017; Liang et al. 2018). A common physiological response of plants to salt stress is chlorophyll degradation. Reduction in chlorophyll leads to a decrease in net photosynthetic rates.

To counteract the osmotic effect, the most significant plant adaptive mechanism is the accumulation of compatible solutes. Plants synthesize and accumulate many kinds of compatible solutes in the cytoplasm to increase their hyperosmotic tolerance against salt-induced water loss from the cells. Compatible solutes such as amino acids (proline and glycine betaine), sugars (glucose, sucrose and trehalose) and sulfonium compounds (choline sulfate) are believed to be involved in the osmotic adjustment mechanisms. This process could mitigate water deficiency in plant cells and protect membrane dehydration (Yildiztugay et al. 2014). It has been widely reported that proline, trehalose and glycine betaine are involved in abiotic stress tolerance in many plant species (Rady et al. 2019; Kosar et al. 2020; Sofy et al. 2020).

To cope with ion toxicity, many ion channels and transporter genes are involved in maintaining ion homeostasis in plant cells. The mechanisms of salt tolerance involved in ion homeostasis involve (i) decreasing net sodium influx by improved selectivity of transport systems, (ii) maintaining K⁺ concentration by reducing efflux and stimulating influx, (iii) maintaining turgor by accumulation of inorganic ions, mainly K⁺ and Na⁺ inside vacuoles and (iv) osmotic adjustment in the cytosol by increased K⁺ concentrations or synthesis of compatible solutes (Munns and Tester 2008; Gálvez et al. 2012). Moreover, exclusion of excess cytoplasmic Na⁺ via salt overly sensitive (SOS) signaling pathway is a significant defense mechanism for several plants. When plants are exposed to salt stress, excessive Na⁺ and high osmolarity are detected by plasma membrane sensors resulting in (i) increased cytosolic calcium ions (Ca²⁺), (ii) an increased Ca²⁺ binding SOS3, (SOS3, salt overly sensitive 3, EF-hand type calcium binding protein) leading to (iii) activation of SOS2 (salt overly sensitive 2); serine/threonine kinase, (iv) the active SOS3–SOS2 protein complex phosphorylates SOS1 (salt overly sensitive 1); plasma membrane Na⁺/H⁺ antiporter, and (v) the activated SOS1 actively excludes Na⁺ ions into the apoplast (Conde et al. 2011). In addition, cytoplasmic Na⁺ ion concentration is controlled by transport proteins located in vacuolar membrane such as vacuolar Na⁺/H⁺ (NHX1) antiporter which pumps Na⁺ ions into the vacuole (Roy et al. 2014).

It is known that salt stress can induce oxidative stress which is caused by reactive oxygen species (ROS) such as superoxide anion (O₂^•−), hydroxyl radical (OH^•) and hydrogen peroxide (H₂O₂). Over-production and accumulation of ROS can damage essential macromolecules in plant cells and disrupt cell redox and energy state (Ahanger et al. 2017). To protect the cells from oxidative stress, plants generate antioxidative defense system, which can be divided into enzymes and non-enzymatic systems. Several antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX) and non-enzymatic antioxidants (glutathione and ascorbate) are capable of detoxifying ROS (Liang et al. 2018).

Among abiotic stress resistance mechanisms, a water-soluble pigment called anthocyanin has been reported to associate with abiotic stress tolerance in plants (Ramakrishna and Ravishankar 2011). Anthocyanin is a colored pigment belonging to flavonoid, found in almost every plant organ (i.e., seeds, leaves, flowers, and fruits) resulting in red, pink, purple, and blue colors (Landi et al. 2013). It has been reported that in many plant species anthocyanin significantly increased under various stress conditions. For instance, Truong et al. (2018) presented that high anthocyanin accumulation involved in salt stress tolerance and nitrate-deficient conditions via promoting nitrate metabolism. Moreover, flavonoid and anthocyanin were reported to enhance drought tolerance by acting as antioxidative agents leading to alleviation of oxidative stress (Nakabayashi et al. 2014).

In the last decade, numerous indigenous and landrace pigmented rice or colored rice varieties in Thailand have been studied for genetic diversity and characterized for unique agronomical traits including their pharmacological properties (Rerkasem et al. 2015; Sivamaruthi et al. 2018). Currently, consumer demand for these types of rice for consumption is increased due to their health promoting phytochemicals (Napasintuwong 2020). However, cultivation area of anthocyanin-containing black rice in the northeast region of Thailand is limited due to saline soil. Since anthocyanin has been reported to be involved in abiotic stress tolerance, understanding the roles of anthocyanin in Thai indigenous pigmented rice under salt stress could fulfill the groundwork for agricultural application and economic foundation.

To address the question whether anthocyanin pigment contributes to salt tolerance, the purple-leaved salt tolerant landrace rice ‘Niew Dam 019’ (ND 019) and the standard salt tolerant green-leaved rice genotype, ‘Pokkali’ (PK) were subjected to salt stress to investigate the role of anthocyanin in response to salinity.

Materials and methods

Plant materials and growth

Green-leaved variety ‘Pokkali’ (PK) and purple-leaved variety ‘Niew Dam 019’ (ND 019) were used in this study. PK is globally well-known for salt-tolerance (Kawasaki et al. 2001). ND 019 is a landrace rice which showed salt tolerance ability (based on the preliminary screening experiment – unpublished data). PK seeds were obtained from Rice Gene Discovery Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. ND 019 seeds were kindly provided by Conservation and Utilization of Thai Indigenous Rice Project, Khon Kaen University, Thailand. For the experiment, rice plants were grown in a greenhouse at the Faculty of Agriculture, Khon Kaen University, Thailand (16°28'29.7" N, 102°48'37.3" E, altitude of 195 m above sea level) under natural sunlight conditions (with the average daily temperature of 30.30 °C, and the mean photoperiod of 12.04 h). The experiment was performed during February to August 2019. Rice seeds of both varieties were germinated in distilled water for 3 d. Further, each germinated seed was placed in a hole in a Styrofoam with nylon net bottom (40 seeds of each genotype), floating on a plastic container containing 6 L of the nutrient solution. The containers were arranged in a completely randomized design. The seedlings were grown for 18 d during which the nutrient solution was replaced every 3 d. After 18 d, the plants were divided into the salt-treated group (nutrient solution supplemented with 150 mM NaCl) and the control, each with four replications. Electrical conductivity of the saline solution in the salt-treated group was maintained daily at 12.5—13.5 dS m⁻¹, and 0.8 – 0.9 dS m⁻¹ for the control. The pH of nutrient solution was maintained daily at 5.0–5.5 (Yoshida 1976; Nounjan et al. 2018). The level of NaCl used in this study was based on our previous experiments (Nounjan et al. 2018). After 5 d of salt stress, the plants were collected for the determination of growth and physiological parameters because it was when the salt-stress symptoms were clearly evident in both genotypes (i.e., older leaves rolled and leaf tips drying). However, for gene expression studies, the seedlings were collected at 0, 1, 2 and 5 d after salt stress. These time points were selected according to the previous report (Kawasaki et al. 2001) which showed that changes in gene expression were induced much earlier than morphological and physiological symptoms. The collected samples were kept at –80 °C and –20 °C until analysis.

Growth and ion measurement

Plant samples were randomly selected 5 d after salt treatment for fresh weight (FW) measurement. Then the samples were dried at 70 °C for 3 d or until weight stabilized and measured for dry weight (DW). Na⁺ and K⁺ in leaves and roots were determined from the dried samples (≈ 0.2 g dried samples) using atomic absorption spectrometry (Model GBC 932 AAA, Cambridge, UK) in the emission mode (Nounjan and Theerakulpisut 2012).

Ion transporter genes expression analysis

The method for gene expression analysis was conducted following Nounjan et al. (2020). Total RNA was extracted from rice leaf tissues (0.1 g) using Trizol reagent (Invitrogen, USA). To remove DNA contamination, RQ1 RNase-Free DNase (Promega, USA) was used to treat the total RNA sample. Total RNA was quantified with NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). Next, first-strand cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit (Fermentas, Lithuania) using 1 μg RNA, 0.5 μg/μL oligo (dT)₁₈ primer and other components according to the manufacturer’s protocol. Finally, quantitative real-time PCR (qRT-PCR) was performed to determine gene expression using LightCycler® 480 instrument (Roche, Germany). The qPCR reactions were carried out in a 96-well plate using LightCycler® 480 SYBR Green I Master (Roche, Germany). Each reaction (20 μL in total) contained cDNA (1:10) as a template and 0.1 μM specific primers (Supporting information T1). PCR condition was set referring to LightCycler® 480 instrument manual. Relative expression level of the target gene was normalized with OsActin and analyzed by 2^−ΔΔCT method (Livak and Schmittgen 2001). The relative expression value of each time point was calculated from three biological replicates.

Determination of chlorophyll content

Total chlorophyll, chlorophyll a and chlorophyll b were extracted from 0.1 g leaf tissue and the absorbance of the extracts were measured at 645 and 663 nm using a spectrophotometer. The chlorophyll contents were calculated using equations given by Arnon (1949) and Larkunthod et al. (2018).

Determination of relative water content, osmotic potential, osmotic adjustment, and compatible solutes

A fully expanded leaf was collected for the determination of relative water content (RWC) and osmotic potential (OP) following the methods described by Larkunthod et al. (2018). The middle zone (2 cm in length) of the leaf blade was used immediately for measurement of RWC. The remaining parts of the leaf blade was frozen in liquid nitrogen and stored at −80 °C, and processed later for measurement of OP. The RWC and OP values were then used to calculate osmotic adjustment (OA).

Proline content was estimated according to Larkunthod et al. (2018) based on Bates et al. (1973) with slight modifications using 0.05 g leaf samples. Proline content in each sample was calculated referring to L-proline standard curve. For determination of sugar, leaf sample (0.05 g) was extracted with 5 mL 80% ethanol at 80 °C, repeated 3 times, and then the extracts were pooled for further analysis. Next, activated carbon was added to eliminate green pigments (Liu et al. 2015). After centrifugation, the clear supernatant was used for determining total sugar by the anthrone method and reducing sugar by the dinitrosalicylic (DNS) method following the protocols of Nounjan et al. (2018) which were modified from Dubey and Singh (1999) and Miller (1959), respectively. Sugar content in each sample was calculated referring to D-glucose standard curve.

Anthocyanin content, total phenolic content, and free radical-scavenging activity

Leaf samples (0.1 g) were used to determine anthocyanin referring to the method of Chunthaburee et al. (2016) modified from Abdel-Aal and Hucl (1999). Anthocyanin content was calculated and expressed as mg g⁻¹ FW.

In order to measure total phenolic content and free radical-scavenging activity which is believed to be found in tissues containing high anthocyanin, the same leaf extract that was used for determining anthocyanin was processed to estimate those two parameters. For total phenolic content, the reaction containing 100 µL of leaf extract and 600 µL of 1:10 diluted Folin-Ciocalteu’s reagent (Sigma-Aldrich, USA) was kept in the dark for 40 min, then the absorbance was measured at 760 nm. The total phenolic content was calculated based on the standard curve of gallic acid (0–20 μg), and expressed as mg gallic acid equivalent g⁻¹ FW (Singleton et al. 1999).

The DDPH (2, 2-diphenyl-1-picrylhydrazyl) scavenging activity was chosen as a representative for free radical-scavenging activity determined in this study. The methods described by Brand-Williams et al. (1995) and Landi et al. (2013) were followed with some modifications. The DDPH activity was expressed as mg Trolox equivalent g⁻¹ FW.

Electrolyte leakage and malondialdehyde

Freshly collected leaf was used to measure electrolyte leakage (EL), small segments of leaf blades (6 segments, approximately 1 cm each) were immersed in 10 mL deionized water for 12 h at room temperature. The first reading of electrical conductivity (EC1) was recorded using a conductivity meter (Gondo, PL-700PC, Taiwan). The tissues were then boiled for 20 min, electrical conductivity after cooling (EC2) was measured, and EL was calculated using the formula: EL (%) = (EC1/EC2) × 100.

For malondialdehyde (MDA), the method described by Hodges et al. (1999) was used with minor modification. Briefly, extraction of each sample was prepared by grinding 0.1 g of leaf tissue with 1 mL 0.1% trichloroacetic acid (TCA), followed by centrifugation at 12,000 g for 10 min. The supernatant was divided into 2 sets of 0.5 mL each. The first set was mixed with 2 mL 0.5% thiobarbituric acid (TBA) in 20% TCA whereas the other was added with 2 mL 20% TCA. The reaction mixtures were then incubated at 95 °C for 25 min. After stopping the reaction in an ice bath, the samples were centrifuged at 10,000 g for 5 min. Absorbance of the clear supernatant was read at 440, 532 and 600 nm using a spectrophotometer. The MDA content was calculated by the formula described by Hodges et al. (1999) and Jacob et al. (2020).

Statistical analysis

The data were subjected to one-way analysis of variance (ANOVA) using SPSS ver. 20 (IBM SPSS Statistics, USA) followed by Duncan’s Multiple Range Tests (DMRT). The differences between means were considered significant at P ≤ 0.05.

Results

Seedling growth and ion contents

Salt stress induced a decline in fresh weight (FW) and dry weight (DW) of both rice genotypes. FW of PK and ND 019 was reduced by 39 and 41%, respectively when compared to those of control plants (Fig. 1a). DW was also decreased (decreased by 42 and 37% in PK and ND 019, respectively) (Fig. 1b). It was noted that, PK plants exhibited higher FW and DW than ND 019 in both control and salt stress conditions.

Fig. 1.

Fresh weight (a) and dry weight (b) of rice plants under control (dark bars) and salt stress (light bars; 150 mM NaCl) for 5 d. All the values are means of four replicates ± SE. One-way ANOVA significant at P ≤ 0.05. Means with different letters above the bars indicate significantly different values (P ≤ 0.05, Duncan’s Multiple Range Test)

The content of Na⁺ in roots and shoots (leaves) of rice grown under control conditions was not significantly different between both rice genotypes. Salinity induces a large amount of Na⁺ accumulation in both tissues. In roots, Na⁺ in PK and ND 019 increased approximately by 3- and 4-fold, respectively (Fig. 2a). In leaves, Na⁺ content increased by 12- and 14-fold in PK and ND 019, respectively (Fig. 2b) as compared to those of control groups. No significant differences in Na⁺ accumulation was found in roots between salt-stressed PK and salt-stressed ND 019 while ND 019 accumulated higher Na⁺ in leaves than salt-stressed PK (Fig. 2a-b). For K⁺, roots of ND 019 accumulated higher K⁺ than PK under non-stress conditions. A decrease in K⁺ content was observed in both genotypes in response to salt stress. There was 45% reduction in K⁺ content of ND 019 and 31% reduction was found in PK (Fig. 2c). In leaves, K⁺ content of non-treated PK and ND 019 did not show any significant differences. When plants were grown under NaCl, K⁺ content slightly decreased in PK (12% compared to those of control groups) but this decrease was not significant. In contrast, K⁺ content of salt-treated ND 019 was significantly decreased under salt stress (28% compared to those of control groups) (Fig. 2d). Similar results of Na⁺ accumulation pattern was observed in Na⁺/K⁺ in roots and leaves of both plants grown with and without NaCl applications. Salt stress increased Na⁺/K⁺ by 5- and 6-fold in roots of PK and ND 019, respectively with respect to controls (Fig. 2e). Higher increase in Na⁺/K⁺ during salt treatment was observed in ND 019 leaves (17-fold) than PK (14-fold) (Fig. 2f).

Fig. 2.

Na⁺ ions in root (a) and shoot (b), K⁺ ions in root (c) and shoot (d), Na⁺/K⁺ ions in root (e) and shoot (f) of rice plants under control (dark bars) and salt stress (light bars; 150 mM NaCl) for 5 d. All the values are means of four replicates ± SE. One-way ANOVA significant at P ≤ 0.05. Means with different letters above the bars indicate significantly different values (P ≤ 0.05, Duncan’s Multiple Range Test). Expression of ion transporter genes (g) SOS3, (h) SOS2, (i) SOS1, (j) NHX1 of rice plants after 0, 1, 2 and 5 d of salt treatment. The histogram shows relative abundance of mRNA for each gene under salt treatment after normalization with OsActin. Values are means of three replications ± SE. For each genotype, bars with different letters indicate that relative gene expression levels are significantly different (P ≤ 0.05) among days after salt treatment according to DMRT

To compare ion transporter gene expression levels between green and purple-leaved rice, three genes in the SOS pathway and one NHX gene were monitored after 0, 1, 2 and 5 d salt treatment using qPCR. SOS3 and SOS1 were significantly up-regulated after PK plants were exposed to salt stress for 1 d (approximately threefold). The transcriptional level of SOS3 was downregulated at 2 d whereas SOS1 expression was gradually decreased. At 5 d, those two genes showed a significant increase of about twofold (Fig. 2g and i). For SOS2, the expression level of SOS2 remained the same during salt treatment (Fig. 2h). For ND 019, no significant change was observed in SOS3 expression during stress period whereas the expression of SOS2 and SOS1 was up-regulated 2- and 4-fold, respectively after 1 d stress period. At d 2, expressions of those genes were decreased (but not significant for SOS2). At 5 d, there was 4- and 3-fold increase in SOS2 and SOS1 expression level (Fig. 2h-i). The expression pattern of NHX1 in both PK and ND 019 was similar. After initial phase of salt application until 2 d, NHX1 transcription did not change. At 5 d after salt treatment, NHX1 was significantly up-regulated to threefold in each genotype (Fig. 2j).

Relative water content, osmotic potential, and osmotic adjustment

Salt stress negatively affected water status in plant cells. Relative water content (RWC) of PK and ND 019 showed similar response when plants were treated with NaCl. RWC was significantly decreased in both genotypes (approximately by 9 and 6% reduction in PK and ND 019, respectively) (Fig. 3a). Under salt stress, osmotic potential (OP) was found to be significantly decreased in PK and ND 019 by 11 and 29%, respectively. However, OP in PK was higher than ND 019 (Fig. 3b). For osmotic adjustment (OA), ND 019 exhibited significantly higher OA than PK (Fig. 3c).

Fig. 3.

Relative water content (a), osmotic potential (b) and osmotic adjustment (c) of rice plants under control (dark bars) and salt stress (light bars; 150 mM NaCl) for 5 d. All the values are means of four replicates ± SE. One-way ANOVA significant at P ≤ 0.05. Means with different letters above the bars indicate significantly different values (P ≤ 0.05, Duncan’s Multiple Range Test)

Osmolytes accumulation

Proline content in ND 019 was higher than PK in the control and salt stress conditions. Under salt stress conditions marked increases in proline were found in both genotypes (2.9- and 3.7-fold increase in PK and ND 019, respectively when compared to those of non-treated groups) (Fig. 4a). Similar trends were observed for total and reducing sugar contents which were significantly higher in ND 019 than PK in both control and salt stress conditions. Total and reducing sugar increased by 20 and 22%, respectively in PK and 32 and 34% in ND 019 compared to those of control groups (Fig. 4b-c).

Fig. 4.

Proline content (a), total sugar (b) and reducing sugar (c) of rice plants under control (dark bars) and salt stress (light bars; 150 mM NaCl) for 5 d. All the values are means of four replicates ± SE. One-way ANOVA significant at P ≤ 0.05. Means with different letters above the bars indicate significantly different values (P ≤ 0.05, Duncan’s Multiple Range Test)

Total phenolic content, anthocyanin and DDPH scavenging activity

The results showed that total phenolic content in the purple-leaved rice, ND 019 was markedly higher than the green-leaved rice, PK in both control and salt stress conditions. Total phenolic content did not significantly change in PK with NaCl treatment whereas 41% reduction of total phenolic content was observed in ND 019 (Fig. 5a). For anthocyanin content, PK showed no significant reduction under salt stress while that in ND 019 significantly decreased (decreased by 46%) (Fig. 5b). The DDPH activities were similar in both PK and ND 019, and no significant salinity induced changes were observed (Fig. 5c).

Fig. 5.

Total phenolic content (a), anthocyanin content (b) and DDPH activity (c) of rice plants under control (dark bars) and salt stress (light bars; 150 mM NaCl) for 5 d. All the values are means of four replicates ± SE. One-way ANOVA significant at P ≤ 0.05. Means with different letters above the bars indicate significantly different values (P ≤ 0.05, Duncan’s Multiple Range Test)

Electrolyte leakage and malondialdehyde content

Under non-stress conditions, ND 019 exhibited higher EL than PK plants. After salt treatment, a marked increase in EL was observed in both PK and ND 019 (an increase of 5.8 folds in PK and 3.5 folds in ND 019). However, there was no significant difference in EL value between stressed plants of the two genotypes (Fig. 6a). The opposite finding was found for MDA. PK and ND 019 under control conditions had similar amount of MDA, but after salt treatment ND 019 accumulated significantly higher MDA than PK. MDA increased by 42% and 119% in PK and ND 019, respectively (Fig. 6b).

Fig. 6.

EL (a) and MDA content (b) of rice plants under control (dark bars) and salt stress (light bars; 150 mM NaCl) for 5 d. All the values are means of four replicates ± SE. One-way ANOVA significant at P ≤ 0.05. Means with different letters above the bars indicate significantly different values (P ≤ 0.05, Duncan’s Multiple Range Test)

Chlorophyll contents

There were no significant differences observed in chlorophyll content between PK and ND 019 plants grown under normal conditions (Fig. 7a-c). After salt treatment, a slight decline (non-significant) in chlorophyll a and total chlorophyll was observed in PK plants while a slight increase in chlorophyll b was found (Fig. 7a-c). On the other hand, significant increases in chlorophyll a, b and total chlorophyll content were noted in ND 019 when plants were exposed to salt stress. Chlorophyll a, b and total chlorophyll content of ND 019 increased by 18, 27 and 20%, respectively compared to those of control groups (Fig. 7a-c). Chlorophyll a:b ratio in both genotypes decreased in response to salt stress. The higher reduction was found in ND 019 followed by PK (decrease by 11 and 9%, respectively) (Fig. 7d).

Fig. 7.

Chlorophyll a (a), Chlorophyll b (b), total chlorophyll (c) and Chlorophyll a:b (d) of rice plants under control (dark bars) and salt stress (light bars; 150 mM NaCl) for 5 d. All the values are means of four replicates ± SE. One-way ANOVA significant at P ≤ 0.05. Means with different letters above the bars indicate significantly different values (P ≤ 0.05, Duncan’s Multiple Range Test)

Discussion

Green- and purple-leaved rice showed similar salt stress response on growth, despite different patterns of ion accumulation and ion transporter genes expression

Different abiotic stresses including salt impose deleterious effects on metabolic functions of plants leading to growth reductions. Although PK plants were more robust than ND 019, as shown in the difference in FW under the controls, both genotypes were similarly affected by salt stress showing similar percent reduction in fresh weight (FW) (Fig. 1a). Nevertheless, dry weight (DW) of salt-stressed ND019 was less decreased than salt-stressed PK (Fig. 1b) compared with the respective controls suggesting that ND 019 was relatively tolerant of salt stress. Numerous experiments pointed out that salt tolerant genotypes suffered less growth reduction in comparison to the susceptible ones (Cheng et al. 2018; Bertazzini et al. 2018; Akbari et al. 2018; Nounjan et al. 2018). In addition, similar reduction in growth percentage of both rice genotypes could indicate similar response under salt stress. It could be proposed from this finding that ND 019, a local Thai landrace variety, had similar level of salt tolerance at seedling stage to the standard salt-tolerant PK.

Under salt stress, an increase in Na⁺ uptake was observed in leaves and roots of both PK and ND 019. High accumulation Na⁺ causes inhibition in K⁺ uptake. As a consequence, high Na⁺/K⁺ ratio was detected when plants were supplemented with NaCl. In roots, both rice genotypes had similar level of Na⁺ and K⁺ contents (Fig. 2a, c). Thus, there was no significant difference in Na⁺/K⁺ ratio in roots of those cultivars (Fig. 2e). The ability to maintain root K⁺ under salt stress has been reported to be associated with tissue tolerance which is one of the major salt tolerance mechanisms (Adem et al. 2014). These findings support their good performance under salt stress indicating the high level of salt tolerance ability probably due to active exclusion of salt by root tissues (Chen et al. 2018). On the other hand, leaves of ND 019 accumulated higher Na⁺ than PK while K⁺ was markedly lower resulting in ND 019 having significantly higher Na⁺/K⁺ than PK (Fig. 2b, d and f). These findings indicate that salt-tolerant PK had better root-to-shoot Na+ exclusion mechanism than ND 019 as evidenced by lower Na+ accumulation in leaves compared to ND 019 despite the fact that they accumulated the same level of Na⁺ in roots (Fig. 2a). Regulation of Na⁺ uptake at roots and translocating less Na⁺ to shoots and leaves meanwhile gathering more K⁺ in every part of the tissues was a characteristic of salt-tolerant rice cultivars (Kavitha et al. 2012).

The SOS pathway is one of the most crucial mechanisms to regulate ion homeostasis under salt stress (Conde et al. 2011). The results showed that there were different expression patterns among SOS3, SOS2 and SOS1 between PK and ND 019 (Fig. 2g-i). Induction of SOS3 in PK was dramatically activated after 24 h of the treatment whereas its expression level was slightly increased, although not significantly in ND 019 at the same point of time (Fig. 2g). SOS3 is the first gene (protein) triggered in the pathway, highly responsive to sense salt stress in PK indicating a faster response to Na⁺ toxicity than ND 019. In this study, the relative expression levels of SOS2 under salt stress seemed to be lower than those of SOS3 and SOS1. However, it was possible that SOS2 protein was naturally present in higher abundance, so its transcripts remained unchanged. Recently, it was reported that more than 86 proteins were naturally more abundant in PK under non-stress condition compared to the salt sensitive IR64. Therefore, PK rice seedlings are primed with high concentrations of proteins in order to face salt stress conditions (Lakra et al. 2019). Among genes functioning in the SOS pathway, SOS1 is the major transporter gene functioning as an anti-transporter responsible for Na⁺ export from cytosol to apoplast (Yamaguchi et al. 2013; Wu 2018). Similar transcription pattern of SOS1 was observed in PK and ND 019. Upregulation of SOS1 in response to initial period of NaCl stress and continuing strong expression until d 5 after the treatment suggested high potential for Na⁺ extrusion. Stronger transcriptional level of SOS1 during salt stress was found in superior salt-tolerant than salt-sensitive genotypes (Chakraborty et al. 2012; Liu et al. 2015; El Mahi et al. 2019). For the SOS pathway, the activation of SOS3 gene when sensing excess Na⁺ could be the key step for controlling Na⁺ accumulation in the leaves of PK as evidenced by the rapid upregulation within 1 d after salt stress (Fig. 2g). Faster sensing of Na⁺ in leaves due to SOS3 function and significant upregulation of SOS3 at 5 d after salt treatment could be associated with low Na⁺ accumulation, resulting in lower Na⁺/K⁺ in PK as compared to that in ND 019 in which SOS3 expression remained stable (Fig. 2b and f).

Sequestration of Na⁺ into vacuoles is a part of mechanisms for reducing Na⁺ content in plant cytosol (Conde et al. 2011). At the first stage of salt treatment up to 48 h, no change of NHX1 expression in leaves of PK and ND 019 was detected. The abundance of NHX1 transcripts in both genotypes was noted after 5 d of salt treatment (Fig. 2j). Compartmentation of Na⁺ into vacuoles by NHX1 might be late response to Na⁺ toxicity compared to the exclusion of Na⁺ to the apoplast by the action of SOS1 which was induced in the first 24 h of salt stress. Like SOS1, upregulation of NHX1 which was reported to be activated by SOS2 (Conde et al. 2011), also contributed to an enhancement of plant salt tolerance by sequestering toxic Na⁺ into vacuoles (Wu 2018). High transcript levels of NHX1 in PK and ND 019 in response to salt stress could be implied that both genotypes had high efficiency in sequestration of Na⁺ into vacuoles. The difference in the regulation of Na⁺ ion transporter genes via SOS signaling pathway in the leaves of salt-stressed PK and ND 019 is illustrated in Fig. 8.

Fig. 8.

Illustrated model comparing the differences, between PK and ND 019 leaves, in Na⁺ stress response of genes encoding the ion transporters (SOS1 and NHX1) regulated by SOS2 which is sequentially activated through the signal transduction pathway involving Ca²⁺ as the second messenger and the Ca⁺² binding protein (SOS3). [PK, Pokkali (the green-leaved genotype); ND 019, Niew Dam 019 (the purple-leaved genotype); ↑, upregulation; − , stable expression]

At the cellular level, the SOS pathway and NHX1 play critical roles in maintaining low cytosolic Na⁺ concentration in plants. In cereals, a family of high-affinity K⁺ transporter (HKT) regulates Na⁺ exclusion from root xylem vessels to minimize shoot Na⁺ accumulation (Mickelbart et al. 2015). The rice OsHKT1;5 gene has been proposed to control Na⁺ flow in the recirculation process from xylem vessel into xylem parenchyma thereby facilitating shoot ion homeostasis by maintaining low shoot Na⁺/K⁺ ratio (Platten et al. 2013). In PK and a halophytic wild rice (Porteresia coarctata), it was reported that the HKT1;5 gene was more highly expressed under high salt concentrations than the salt-sensitive rice, IR29 (Shohan et al. 2019). In this study, the observed low shoot Na⁺/ K⁺ in PK might be attributed to the activity of this ion transporter gene.

Purple-leaved rice showed superior osmotic adjustment to green-leaved rice under salt stress

Water status is a common indicator used for evaluating plant stress response. Stronger tolerant genotypes often show better regulation of water content (indicated by RWC) than susceptible ones under stress conditions indicating higher water availability in plant cells (Adem et al. 2014; Bistgani et al. 2019). There was no significant difference in RWC between PK and ND 019 under salt stress suggesting both genotypes had similar ability to control water status (Fig. 3a). When NaCl was applied, a decrease in osmotic potential was observed accompanied with high amount of osmolyte (proline and sugar) accumulation in both genotypes (Fig. 4). Many reports concluded that built up of proline and sugar in response to salt stress associated with more negative osmotic potential values and plant salt tolerance (Zrig et al. 2016; Akbari et al. 2018). It was clearly seen that under salt treatment ND 019 accumulated higher osmolytes leading to more negative osmotic potential, hence greater potential of the cells to uptake water from external medium with low water potential, than PK. In parallel, occurrence of high osmolytes accumulation, especially in ND 019 could associate with high osmotic adjustment in this genotype (Fig. 3b-c). In addition to providing osmotic adjustments, osmolytes also function in protecting damage to macromolecules. Liang et al. (2013) illustrated the protective role of proline on protein stabilization under osmotic stress. Moreover, osmolytes can play roles in mitigating negative effects of high ion concentrations on enzyme activity (Bohnert and Shen 1999).

In addition, anthocyanin which is found in higher concentration in ND 019 has been reported to function as osmoregulators, and its role is more prominent in red-leaved than green-leaved plants (Zrig et al. 2016). The positive correlation among anthocyanin, osmotic ptential and osmotic adjustment was also pointed out (Zrig et al. 2018). Adaptation to tolerate the osmotic effect caused by salt stress via osmotic adjustment mechanism allowed plants to survive under limited water supply conditions (Parihar et al. 2015). Although PK showed lower osmotic adjustment compared to ND 019, well-maintained water in plants under salt stress may result from less harmful ion toxicity effects due to less Na⁺ accumulation in leaves (Fig. 2b). Therefore, these findings suggested that osmotic adjustment was not the major contribution mechanism against salt stress in green-leaved, PK at seedling stage.

Salt stress induced a decline in total phenolics and anthocyanin in purple-leaved rice in contrast to green-leaved rice

Purple-leaved rice plants had higher total phenolic compounds and anthocyanin than green-leaved in both non-stress and salt stress conditions. Previous studies reported that salt stimulated high production of phenolics and anthocyanin (Truong et al. 2018; Bistgani et al. 2019). However, in this present study salt stress did not induce any significant changes in both compounds in green-leaved rice, PK. This may be because anthocyanin is not a major color pigment accumulated in PK leaves. Conversely, salt stress causes a significant reduction in total phenolic compounds and anthocyanin in purple-leaved rice, ND 019 (Fig. 5a and b). The reduction in anthocyanin may be caused by Na⁺ toxicity due to high accumulation of Na⁺ in ND 019 leaves (Fig. 2b). Changes in anthocyanin content were correlated with the total phenolic content which depended on NaCl concentration and characteristics of plant genotypes (Waśkiewicz et al. 2013; Zrig et al. 2016).

Similar level of DDPH scavenging activity was observed in both green- and purple- leaved rice plants under both non-stress and salt stress conditions (Fig. 5c). Many studies presented that high DDPH activity associated with salt stress tolerance ability in plants (Kaur et al. 2014; Bagues et al. 2019). Phenolic compounds are able to act as non-enzymatic antioxidant molecules (Chen and Raji 2020). Noticeably, reduction in total phenolic content in ND 019 under salt stress did not coincide with DDPH activity (Fig. 5a, c) suggesting that efficiency of antioxidant capacity in ND 019 did not associate with total phenolic content. Sahitya et al. (2018) proposed that total phenolic level did not associate with DDPH activity. Some genotypes might show high level of total phenolics but low DDPH activity depending on plant genotypes, stress treatment conditions and duration time exposure to stress.

Under salt stress conditions, ND 019 showed more damage from oxidative stress than PK plants as evidenced by higher MDA content though both genotypes had similar EL level (Fig. 6). Higher MDA in ND 019 may be caused by significant decrease in total phenolic content leading to less ability to scavenge ROS and higher Na⁺ accumulation in leaves of ND 019 compared to PK (Fig. 2b). However, ND 019 showed good growth performance under salt stress conditions (Fig. 1). From these findings it could be proposed that ND 019 was able to keep the balance between ROS generation and antioxidant defense activities as evidenced by its minimal growth reduction.

In some studies, an increased level of anthocyanin has been reported to contribute to salt stress tolerance (Oh et al. 2011; Chunthaburee et al. 2016). Nevertheless, in the present study, anthocyanin decreased due to salt stress. In another study, salt stress led to an increase in anthocyanin in one tomato cultivar but a decline in the other (Borghesi et al. 2011), suggesting that the relationship between salt stress and anthocyanin production is still inconclusive. From this study, it could be proposed that anthocyanin did not directly contribute to salt resistance in purple-leaved rice, ND 019.

Salt stress did not influence green pigments in green-leaved rice, PK while induced a decline in purple pigment of purple-leaved rice, ND 019

The main role of chlorophyll is involved in the photosynthetic process which is essential for light absorption while anthocyanin provides helpful roles to protect plants from photo-oxidative stress (Hughes et al. 2012). It is known that salt stress induces a decline in chlorophyll biosynthetic process leading to decrease in chlorophyll content. Generally, chlorophyll content in salt-tolerant plant genotypes showed less decrease or better chlorophyll retention than salt-sensitive ones (Chakraborty et al. 2012; Kibria et al. 2017). Chlorophyll a, b and total chlorophyll content of PK remained unchanged under salt stress condition (Fig. 7a-c) suggesting its salt tolerance ability. In case of ND 019, those parameters were increased under salt treatment (Fig. 7a-c). An increase in chlorophyll might be one of the adaptive strategies of plants to survive under unfavorable growth conditions (Shah et al. 2017). Purple-leaved rice, ND 019, has higher anthocyanin than green-leaved rice, PK. Reduction in anthocyanin and high chlorophyll accumulation were observed in ND 019 when salt stress was applied (Fig. 5b, c). With the increased chlorophyll level and decreased anthocyanin content in leaf of ND 019 plant, the leaf finally became green. Ren et al. (2019) pointed out that degradation of anthocyanin resulted from decreased anthocyanin synthesis and increased anthocyanin degradation processes. Accumulation of chlorophyll started when the ratio of anthocyanin/ chlorophyll decreased. A decline in chlorophyll a:b was found in green-leaved, PK, and purple-leaved genotype, ND 019, under salt stress. Nevertheless, PK showed higher chlorophyll a:b than ND 019 (Fig. 7d). Reduction in chlorophyll a:b ratio under salt stress may associate with disintegration of grana stacking (Shu et al. 2012) suggesting adverse effect of salt stress was more pronounced on chlorophyll a:b in ND 019 than PK plants (Fig. 5b). However, in some purple-leaved rice genotypes, the positive correlation between anthocyanin and chlorophyll content were observed when plants were exposed to moderate level of salt stress (100 mM NaCl) for four days (Chutipaijit et al. 2011). This trend was also observed in Fragaria chiloensis when low level of salt stress (60 mM NaCl) was applied (Garriga et al. 2014).

Although ND 019 expressed similar extent of salt-induced growth reduction as that of the standard salt-tolerant PK, physiological responses attributed to salt tolerance obviously differed as summarized in Fig. 9. In PK, the dominant tolerance mechanism involved the more efficient shoot-to-root Na⁺ exclusion and earlier expression of the SOS3 gene which regulates Na⁺ exclusion from leaf cells while the osmotic adjustment was less significant. On the other hand, the purple-leaved ND 019 was less efficient in ion exclusion but accumulated higher compatible solutes leading to a reduction in salt-induced damage due to an efficient osmo-protective mechanism.

Fig. 9.

Diagrammatic representation of differential salt tolerance mechanisms between Pokkali (the green-leaved genotype) and ND019 (the purple-leaved genotype). [= , similar response or comparable quantity; + , more efficient or greater quantity; -, less efficient or lower quantity]

Several reports suggested that high anthocyanin accumulation in purple-leaved plants during salt stress enhanced plant salt tolerance ability. However, our results indicated that anthocyanin did not involve in mitigating adverse effects of salt stress. The protective roles of anthocyanin during salt stress may vary depending on many factors including the level of NaCl, the time interval after salt treatment, genotypes, and the developmental stages of plants. Suggestions for future experiments to better explain the roles of anthocyanin in relation to salt stress protection are an inclusion of more purple-leaved rice cultivars with different levels of salt tolerance, varying NaCl concentrations and duration of stress exposure, and employing integrated ‘omics’ approaches.

Conclusion

Comparisons of salt tolerance responses in two rice genotypes having contrasting leaf color, one having green (PK) and the other purple leaf (ND 019) in relation to their growth, physiological, biochemical and ion transporter genes expression revealed that both genotypes showed comparable levels of salt tolerance. However, there were considerable differences in the mechanisms for salt stress tolerance. The data obtained in this work revealed that the major salt tolerance mechanism in the green-leaved rice, PK, resulted from ion exclusion which prevented high accumulation of toxic Na⁺ in leaves. On the contrary, osmotic adjustment was the main function which mitigated negative effects of salt stress in the purple-leaved rice, ND 019. Superior osmotic adjustment in ND 019 to PK resulted from higher accumulation of compatible solutes. It is interesting to note that salt stress caused anthocyanin reduction in anthocyanin-rich ND 019. Therefore, the roles of anthocyanin in protecting purple-leaved rice from salt stress need to be further investigated.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

SOS

Salat overly sensitive

ROS

Reactive oxygen species

ND 019

Niew dam 019

PK

Pokkali

RWC

Relative water content

OP

Osmotic potential

OA

Osmotic adjustment

Funding

This work was supported by a grant from the National Research Council of Thailand (NRCT) through the Senior Research Scholar Project of Prof. Dr. Piyada Theerakulpisut (Project No. NRCT813/2563). The scholarship under the Postdoctoral Program from Research Affairs and Graduate School, Khon Kaen University (58331) to N.N. was gratefully acknowledged.

Declarations

Conflict of interest

All authors declare no conflict of interest.