1. Introduction
Rice is one of the most widely consumed grains in the world, supplying carbohydrates and nutrients to more than half of the world’s population. The widespread use of rice in food (e.g., cereals, oils, beverages, and flour) and medicine, combined with a growing population, have resulted in massive demand for rice over the decades [
1,
2]. Reduced arable land, climate change, and biotic and abiotic stresses are the major contributors to limited global rice crop production. Salinity is one of the major factors reducing global rice yields [
3]. It is estimated to affect ~20% of cultivated and ~33% of irrigated farmland worldwide, and is predicted to affect more than half of total arable land by 2050 [
4,
5].
Previous reports have found that high salt stress has several negative effects on rice plants, including germination interference, stunted growth, impaired fertility, flowering inhibition, and reduced photosynthetic activity [
1,
6]. Germination and emergence are critical stages in the plant’s life cycle for regulating the plant’s efficacy in utilising available water and nutrients. Perturbation at any of these stages has a significant impact on the plant’s subsequent growth, resulting in a yield penalty [
7]. For instance, a high salt level in plant tissue has been shown to reduce leaf photosynthetic activity and respiration rate, inhibiting overall plant growth [
8]. The reduction of photosynthetic activity of plants under salinity stress can be attributed to several factors: (1) limitations in CO
2 availability due to stomatal closure; (2) loss of function of photosynthetic pigments following degradation and/or oxidative damage; and (3) biochemical modifications [
9,
10]. Thus, prolonged exposure to a saline environment results in the reduction of the concentration and translocation of assimilates across plant organs that are crucial for cell growth and development [
10].
Rice plants able to confer higher growth performance in a saline environment over a period of time are deemed “tolerant” and have higher potential in contributing to food security [
11]. Changes in morpho-physiological traits frequently depend on the regulation of plant molecular and biochemical processes in response to stress. Biochemicals such as sugar alcohols, quaternary ammonium, proline, and tertiary sulfonium modulate critical processes such as osmoregulation, protein stability, membrane permeability maintenance, ROS scavenging, and photosystem association [
12,
13]. Cumulative effects of biochemical alterations under stress help plant adaptation and survival in saline environment, ensuring steady growth while minimising damage caused by osmotic and ionic stress [
13].
An increase in salt level also triggers a series of continuous signals that contribute to the activation or inactivation of particular proteins or genes involved in specific biological processes [
14]. Transcript profiling of different rice varieties by Basu and Roychoudhury [
15] showed that salinity stress triggers upregulated expression of stress-related genes such as SOS3, NHX1, HKT1, PAL, and CHS. These genes encode proteins that play important roles in metabolic pathways and plant tolerance mechanisms against salinity such as transport, detoxification, stress-related hormone biosynthesis energy, and more [
15]. The activation of salt overly sensitive (SOS)-related proteins, for example, modulates the exclusion and sequestration of excess Na
+ in the cell and prevents ionic imbalance [
16,
17]. An increase in the activity of enzymatic antioxidants such as superoxide dismutase (SOD), peroxidase, and catalase (CAT) reduces and prevents the accumulation of reactive oxygen species (ROS), minimising toxicity and damage to cell components [
18].
MR219 (
Oryza sativa L. ssp.
indica) is a commercial Malaysian rice variety that was released in 2001 and is reported to have high yield production, good grain quality, a short maturation period, and resistance to infectious pests such as blast and bacterial blight [
19]. However, MR219 is quite sensitive to environmental changes such as drought and flooding [
20,
21,
22]. Although there is good understanding of the effect of salinity on rice in general, studies on the Malaysian variety are still limited. The purpose of this study is to determine MR219′s response to salt treatment by comparing the morpho-physiological and biochemical changes between MR219 and a salt-tolerant variety, Pokkali, as well as susceptible variety, IR64. The expression levels of genes involved in gamma-aminobutyric acid (GABA) metabolism, ion transport, and ROS detoxification were also investigated to understand plant responses to salinity stress at a transcriptional level. Here, Pokkali and IR64 were used as control varieties due to their remarkably high tolerance level and susceptibility to salinity, respectively, as reported in many previous salinity-related studies [
23,
24,
25,
26].
Rice varieties that show high tolerance to salinity should have better growth performance in terms of shoot height, root length, biomass, leaf area, and number of nodes and leaves, as well as higher chlorophyll and relative water content compared to susceptible varieties. We hypothesized that MR219 could withstand the salinity stress during the vegetative stage, hence contributing to better growth performance traits than that of susceptible variety. The salinity tolerance can be determined by the roles of GABA metabolism, ion transport, and antioxidant activity. Obtaining this information will assist in further understanding the coping mechanism of MR219 to salinity stress.
4. Discussion
The results of germination performance indicate that the MR219 seed has slight tolerance to salt stress and is unaffected by low to moderate salinity levels, though the time required for the seed to germinate appears to be delayed as the salinity level increases. Interestingly, the seed germination rate of MR219 was improved at a low level of salinity, which contrasts with the findings of recent studies on rice seed germination [
32,
33]. It is, however, supported by an earlier study by Panuccio et al. [
34] reporting that low salt treatment speeds up seed germination of quinoa, but not the final germination percentage. As suggested by Zhang et al. [
35], sodium or salt may act as osmoticum whereby the absorption of sodium into seeds may facilitate the uptake of water more rapidly. In contrast, high salinity levels have a significant impact on MR219 seed, completely halting germination and radical emergence. Similar results were also reported by Roy et al. [
36] that high salinity stress treatment reduces the germination rate and percentage of two different Binadhan varieties. Likewise, an increase in salinity concentration caused inhibition of >80% seeds of both tolerant and susceptible rice varieties after 10 days of treatment [
37]. This could be due to metabolic changes such as reduction in K
+ efflux and solute leakage caused by mineral nutrient imbalances under salinity stress [
3,
32]. Meanwhile, Na
+ build-up caused a K
+/Na
+ imbalance, which disrupts many physiological functions of MR219, including germination. Salinity also reduces moisture availability, which is required for embryo activation.
During the germination stage, salinity reduces turgor pressure, which may limit the elongation of cells and cause stunted growth, as evidenced by shoot height and root lengths, which are important morphological parameters for growth and development. Previous research found that young seedlings are more sensitive to salinity than older plants, as plumule growth is greatly affected even when treated with low salt stress [
27,
38]. Osmotic stress, low nutrient availability, and K
+ efflux may inhibit shoot growth, interfering with a variety of growth processes such as enzyme activation, stomatal activity, photosynthesis, sugar transport, protein and starch synthesis [
39,
40]. The plumule’s growth is initially suppressed more than the radicle’s, with plumule length being shorter and the root appearing to grow longer compared to the control at low salinity. This observation is supported by research by Hakim et al. [
27] and Fogliatto et al. [
33], which suggests that the shoot is more susceptible to salinity than the root. At high salinity levels, the growth of radicles and roots was shown to be severely hampered, as opposed to shoots, where the emergence of radicles from most seeds was completely halted. The positive correlation between root growth and salinity at low levels could be attributed to the MR219 adaptive strategy, suggesting that seedlings can still maintain homeostasis under low salinity stress. Moreover, low water potential due to limited water availability could cause root elongation in MR219. In contrast to high salinity levels, the shortening of the MR219 roots may be able to restrict the uptake of Na
+ and Cl
- ions into the plant transport system via root tissues to prevent toxic sodium accumulation, hence the inhibitory effects of salt on root growth.
Growth stalled immediately after salt treatment in MR219 grown in a hydroponic support system. Unlike other salt-tolerant varieties, MR219 is susceptible to salinity even at the late vegetative stage. The reduction in the number of leaves is consistent with the observation in rice and other plant species such as tomato, rosemary, and more [
41,
42,
43]. These reductions indicate that salinity inhibits cell elongation and division, which directly inhibits rice plant vertical growth. The reduction of leaf surface area and the number of leaves could be attributed to the plant’s stress-avoidance mechanism to prevent excessive water loss and support the retention of toxic ions in the root rather than the aerial part of the plant [
44,
45]. Moreover, the inhibitory effects of salinity on leaf growth of different rice varieties have also been reported in previous studies by Kazemi et al., and Siregar et al. [
46,
47]. Salinity also inhibits biomass allocation, resulting in a decrease in plant biomass under salinity stress. This study has found that Pokkali is less affected by salinity than MR219 and IR64, as evidenced by the high plant height, biomass, leaf surface area, number of leaves, and number of nodes under stress. This could be attributed to Pokkali’s efficiency in growth- and development-related biological processes, which may contribute to the plant’s higher growth rate even under normal conditions when compared to MR219 and IR64. Pokkali may be able to adapt to salinity stress better if it has a high photosynthesis rate, efficient carbon utilisation for growth and maintenance, and high antioxidant activity. The root growth of salt-tolerant Pokkali is more sensitive to salinity than the salt-susceptible IR64, as evidenced by the increased shoot-to-root ratio. This observation, however, contradicts the majority of studies, which show that increasing root surface area aids salinity-tolerant plants in retaining toxic ions in the root and controlling their distribution to other parts [
44,
48]. Regardless, the results in this study are supported by Safitri et al. [
49] on similar rice genotypes treated with 120 mM NaCl, where Pokkali showed a 37.9% decrease in root length compared to a 19% decrease in IR64. Plant alternatives, such as limiting soluble salt accumulation in the shoot and delaying the onset of the tolerance threshold, could explain the inhibition of root growth, as suggested in previous studies [
45,
50,
51].
Salinity causes a decrease in water potential, which induces cell dehydration, including that of guard cells on the leaf. This phenomenon causes the stomatal aperture to close during salinity stress, which helps to reduce water loss from the leaf tissues and maintain osmotic homeostasis [
44,
52]. With a decrease in leaf surface area and the number of leaves, the decrease in stomatal conductance due to stomatal closure indirectly reduces the plants’ overall photosynthesis rate and transpiration rate [
53]. Similar results were observed when other plant species were subjected to salinity stress [
10,
54]. As previously suggested [
55,
56], the decrease in stomatal conductance may contribute to plant survival against salinity by maintaining cellular osmotic homeostasis and reducing toxic ion translocation through transpiration flow. Regulation of leaf osmotic potential has an indirect effect on leaf RWC. At the end of treatment, only Pokkali and MR219 managed to retain RWC readings above 70%. This finding suggests that Pokkali and MR219 maintain osmotic homeostasis better than IR64 under salinity stress. Aside from stomatal closure, the decline in gaseous exchange activities could be attributed to the high concentrations of soluble ions (e.g., Na
+ and Cl
−) that damage the thylakoid membrane in the chloroplast [
57].
The increased toxicity caused by ROS accumulation damages plant tissues and organelles, disrupting biological processes critical for survival, including photosynthesis and carbon metabolism. The decrease in chlorophyll content in the leaf sample could be attributed to ROS toxicity. In this study, the early and greatest reduction in chlorophyll observed in IR64 from days 6 to 10 may contribute to the photosynthesis rate reduction under stress. Despite the decrease in photosynthesis rate, there were no significant changes in chlorophyll content in Pokkali and, to a lesser extent, MR219. In this case, the decrease in photosynthesis rate in Pokkali and MR219 could be attributed to a decrease in stomatal conductance and transpiration rate or inhibition of related biochemical processes such as carbon dioxide assimilation [
58]. Salinity also affects the photosynthates allocation within plants, which influences plant growth and maintenance under stress. Photosynthates may act as osmolytes to combat osmotic pressure, allowing the plant to tolerate salinity [
59,
60]. This may explain Pokkali’s superior growth performance in terms of morpho-physiological traits and the gaseous exchange rate when compared to MR219 and IR64 under normal and salinity-treated conditions.
To understand the biological mechanism underlying the changes in plants morpho-physiology under salinity, we also investigated the expression of genes involved in plant tolerance mechanisms against salinity. Among the wide range of pathways and mechanisms involved in plant responses and tolerance against salinity, GABA metabolism, ion transport, and detoxification are considered key mechanisms and have been highly studied among researchers [
61,
62,
63]. It was found that the GABA shunt may provide an alternative carbon source in the TCA cycle when part of the TCA cycle is perturbed or downregulated during salinity stress [
64]. Previous research has also shown that salinity-tolerant wheat plants show upregulation of transcript encoding components of the GABA shunt pathway upon salinity treatment [
65]. Moreover, according to Shetewy et al. [
62], exogenous application of GABA improves rice growth performance and tolerance against salinity stress. To the best of our knowledge, however, reports on the activity of components involved in the GABA shunt pathway, specifically in rice, are still scarce and have yet to be carried out.
In this study, treated rice plants showed increases in
GABA-T1,
GAD2, and
SSADH expression at least once during the treatment period in MR219, Pokkali, and IR64. Pokkali and MR219, specifically, managed to maintain their high expression levels of those genes at the end of treatment, despite a slight downregulation during the stress. In contrast, expression in IR64 plummeted significantly after 10 days of treatment, suggesting that the anaplerotic function of the GABA shunt is important in conferring salinity stress tolerance in rice. This may also explain the low level of tolerance in IR64, which may be attributed to the low energy level supporting the stress-tolerance mechanism for survival under stress. This notion is in agreement with previous research, which found a decrease in salinity tolerance in plants with mutated genes encoding GABA-shunt pathway components [
66,
67]. Aside from that, salinity enhances the expression of genes involved in ion transport.
SOS2 expression increased significantly in MR219, Pokkali, and IR64, especially after 2 days of treatment. This rapid response demonstrates rice plants’ critical response to maintaining ionic homeostasis under stress. The expression, however, decreased over time, especially in IR64. This could be due to degradation or damage by ROS, which affects the ion transport activities in IR64. As demonstrated by Pokkali, the ability to maintain high
SOS2 expression throughout stress reflects plant tolerance to salinity
There was a significant increase in the expression of genes encoding enzymatic antioxidants such as CuZnSOD, MnSOD, and CAT in Pokkali, MR219, and IR64. These antioxidants are essential for detoxification and protection against oxidative damage caused by ROS accumulation [
68,
69].
CuZnSOD1 overexpression under stress in all varieties suggests ROS scavenging mechanisms in the cytosol, chloroplast, and peroxisome [
70,
71]. Pokkali maintains high CuZnSOD activities throughout the treatment period, whereas MR219 and IR64 have
CuZnSOD1 expression downregulated as the treatment progresses. This observation corresponds to Pokkali’s high photosynthesis rate and chlorophyll content, as previously discussed. Meanwhile,
MnSOD1 expression increased in all varieties, indicating an improvement in the ROS scavenging mechanism in the mitochondria and peroxisomes [
72]. The correlation between increased activity and/or expression of these genes with tolerance to salinity is discussed in previous studies [
73,
74,
75]. Besides SOD, salinity affects the expression of genes encoding CAT. According to Ighodaro and Akinloye [
76] and Poli et al. [
77], SOD activity is positively correlated with CAT, which is involved in the conversion of H
2O
2 into water. There was an increase in
CAT1 expression in MR219, Pokkali, and IR64 under stress. This suggests that the plant response improves the scavenging mechanism for photorespiratory products such as H
2O
2 [
78]. In contrast to
CuZnSOD1 and
MnSOD1 expression, transcription of
CAT1 decreases with treatment duration for all varieties. This could be attributed to the plant’s inability to counter the damaging effects of excess H
2O
2 produced by photorespiration or even SOD action. It also indicates that CAT activities are independent of SODs and differ among plant species. Overall, increased antioxidant activities improve the ability of plants to limit oxidative damage, resulting in salinity tolerance.
This study provides a focused comparative study on effects of salinity towards germination, morpho-physiological traits, and stress-related gene expression of MR219 with salinity-tolerant Pokkali and salinity-susceptible IR64 varieties. Compared to previous studies, the data collected show changes in the parameters at different time-points throughout the treatment period, allowing us to identify the plant responses against osmotic and ionic stress. It also enables us to determine the changes in the expression of selected stress-related genes encoding components that may involve in MR219 tolerance activity against salinity, for example components that involved GABA shunt pathways, which is novel in rice studies. Furthermore, the changes in morphological traits and transcriptome analysis of MR219, Pokkali, and IR64 in response to salinity will provide a baseline for comparison in future investigations through other omics platforms such as proteomics and metabolomics. This allows us to identify the target gene/protein/metabolite involved in plant responses and salinity tolerance mechanisms, which can then be followed up with functional studies. This is significant since MR219 is one of the elite varieties that has been widely distributed in Malaysia. As a result, any research aimed at improving the performance of local rice crops is critical.