The Mekong Delta is one of three deltas severely affected by climate change, which damages the production of rice in this region through drought and saltwater intrusion in the period 2000-2050 (Ty et al. 2015). Drought and salt stress affect rice physiologically and biochemically. ROS (reactive oxygen species) molecules accumulate in rice plants during drought and salt stress, altering protein structure, lipids, DNA, and metabolic activities. Rice also experiences stress-induced pigment losses, photosynthetic impairments, and negatively impacted accumulation, metabolism, and homeostasis (Farooq et al. 2019; Hussain et al. 2017; Luo 2010), which impacts rice development and grain size, leading to lower rice yields (Hussain et al. 2017). While drought mainly affects rice water uptake and stomatal closure (Farooq et al. 2019), salt stress causes Na⁺ ion poisoning by affecting the plant’s K⁺/Na⁺ ratio and other essential ion uptake (Hussain et al. 2017). In addition to avoiding the impact of climate change on rice production, developing infrastructure for managing and using water resources, evaluating, recreating, and selecting tolerant varieties can help rice farmers cope with climate change (Luo 2010).

Molecular markers aid breeding by identifying phenotypic traits through genotype (Ly et al. 2020). Different studies on rice have identified multiples molecular markers including RFLP (Restriction Fragment Length Polymorphism), AFLP (Amplified Fragment Length Polymorphism), SSR (Simple Sequence Repeat), and SNP (Single Nucleotide Polymorphism) related to drought and salt tolerance (Waziri et al. 2016; Zheng et al. 2008). These markers are associated with drought and salt tolerance in seedlings, membrane stability, biology, penetration depth, root physiology, plant shape, cell control, and osmosis. Verma’s study found 147 polymorphic alleles on 114 rice cultivars using 65 SSR markers (Verma et al. 2019). In Vietnam the SSR markers are being used to breed drought- and salt-tolerant rice (Nguyen et al. 2016).

Tai Nguyen (TN) has been a popular rice cultivar in the Mekong Delta for a long time. This cultivar is known for its quality grains with high opacity, soft texture, spongy texture, and sweet taste (Thu and Loc 2020), and was selected for its adaptation traits to the local conditions. As climate change is causing salt intrusion, widely affecting the rice culture in the Mekong Delta, the good salt tolerance of the TN cultivar suggests its potential for rice production in the region (Huynh et al. 2019). The molecular and biochemical mechanisms behind the tolerance of this rice cultivar were yet to be investigated. In this study, we focused on the effects of drought and salt conditions on the growth and antioxidant enzyme activities of TN rice seedlings, a stage that is strongly sensitive to both drought and salt stress (Luo 2010). We also investigated the presence of some important rice SSR markers associated with drought and salt tolerance.

Plant growth conditions and stress induction

The Tai Nguyen (TN) rice cultivar is taken from the specialized cultivation area of My Le commune (Can Duoc district, Long An province, Vietnam). Oryza sativa spp. Japonica cv. Nipponbare (Nipponbare) - a drought- and salinity-sensitive cultivar (Jiang et al. 2013; Murshidul and Kobata 1998) was provided by Dr. Anne-Aliénor Véry, CNRS, SupAgro, INRAE, Montpellier, France. Seeds were soaked in warm water (60°C) for 48 hours, with water renewed every 6 hours, and germinated in a wet cloth after 2 days (Islam et al. 2018). After germination, seedlings were grown in 9-cm petri plates containing Yoshida solution. For drought stress experiments, Yoshida solution supplemented with polyethylene glycol 6000 (PEG 6000) (Bio Basic Inc, Canada), (0%, 5%, 10%, 15%, 20%) was applied to 5-day-old seedlings (25 seeds/plate, 3 plates/treatment for physiology analysis and 5 plates/treatment for biochemical analysis). For salt stress, Yoshida solution supplemented with NaCl (0, 30, 60, 90, 120 mM) induced seeds right after they germinated. For both stresses, seedlings were exposed to stress treatments for 7 days. The growing process and induction were done in a growth chamber (funded by TWAS Grant Award 16-142 RG/BIO/AS_I-FR 3240293339) at 30°C, 70-80% humidity, and photoperiod 12 hours. Plates were randomly placed within the growth chamber and their positions were randomly swapped every day to avoid bias due to uneven environmental conditions such as air flow or light intensity. For physiological analysis, five random plants per plate were analyzed to obtain the mean values for each plate, and the data were presented as a mean of three plates (mean ± SE). For biochemical analysis, one sample per plate was randomly chosen for analysis, and the data were presented as a mean of five samples (mean ± SD).

Biochemical analysis of rice seedlings under drought and salt stresses

Chlorophyll and carotenoid quantification followed the methods from Su et al. (2010). Leaves were ground in 6 mL of cold 96% Ethanol and incubated at 4°C for 1 hour. The residue-free liquid was examined for absorbance at 480, 645, and 663 nm (Su et al. 2010). All absorbance measurements in this study were performed using spectrophotometer (Thermo Scientific™ Multiskan SkyHigh).

Proline quantification method from Ábrahám et al. (2010). The shoots were homogenized in 3% sulfosalicylic acid. A 1:1:1 volume ratio of 2.5% ninhydrin, acetic acid and decanted extract was reacted in 100°C for 30 minutes. Toluene was vortexed 1:1 with the reaction mixture. Absorption at 520 nm was measured from the supernatant (Ábrahám et al. 2010).

Protein extraction and determination of antioxidant enzyme activity: 0.2 g of fresh sample was homogenized using liquid nitrogen and 2 ml of 0.1 M phosphate buffer pH 7.5, 3 mM EDTA, and 0.5% PVP. Protein content was measured using the Bradford reagent and a BSA standard curve. Nitrotetrazolium blue chloride-NBT was used to quantify the superoxide dismutase (SOD) enzyme activity following Spitz and Oberley (1989). A 200 µL reaction solution contained phosphate buffer pH 7 (0.1 M), methionine 14 µM, EDTA 0.1 µM, NBT 74 µM, riboflavin 2 µM, and 10 µL enzyme extraction. Absorbance was measured at 560 nm. The reaction mixture contained 200 µL of 0.1 M phosphate buffer pH 7, 0.5 µM vitamin C, 0.1 µM H₂O₂, and 10 µL enzyme extraction at 290 nm every 2 min to evaluate ascorbate peroxidase (APX) enzyme activity. Guaiacol peroxidase (GPX) enzyme activity was measured at 420 nm in a 200 µL reaction mixture of 3 µM guaiacol, 13 µM H₂O₂, 0.1 M phosphate buffer, and 5 µL enzyme extract (Mishra et al. 2013; Spitz and Oberley 1989; Vighi et al. 2017).

Evaluation of stress-related SSR markers

DNA was extracted following the CTAB method of Doyle (Doyle 1991). SSR marker primers for drought including RM223, RM228, RM164, RM263 and salinity including RM23, RM493, RM562, RM3412 were obtained from Gramene database (Tello-Ruiz et al. 2022). PCR reactions were performed according to the instructions of the GoTaq Green Master Mix kit (Promega Corp, USA). Results were determined by electrophoresis on 3% Agarose at 90 V for 30 minutes with a 1kb HyperLadder (Meridian Bioscience InC, UK). TN genome data was acquired from NCBI, SRA accession number SRR11278883, and was cleansed (Trimmomatic) and aligned (BWA) to Nipponbare genome. The alignment was visualized and manually adjusted with Geneious Prime 2025.0.2 (free trial) (Bolger et al. 2014; Jung and Han 2022).

Statistical method

Statistical difference was determined by ANOVA one-way (P < 0.05) and followed by Duncan post hoc test on SPSS version 20 software.

TN rice cultivar showed strong drought tolerance

When grown in 20% PEG, Nipponbare seedlings died after 7 days (Fig. 1A). Most leaves were rolled and dried, and all roots turned brown. In the control condition, the plants had fresh green foliage, growing upright, and white roots.

Figure 1. Nipponbare rice seedling growth and root development after 7 days of exposure to drought or salinity stress. (A) Shoot (top panel) and root (bottom panel) development under drought stress: left, 0% PEG (control); right, 20% PEG. (B) shoot (top panel) and root (bottom panel) development under salinity stress: left, 0 mM NaCl (control); right, 120 mM NaCl. The scale bar represents 2 cm

For the TN cultivar, upon drought stress treatments, a continuous decline in shoot growth indicators with the increasing PEG content from 0% to 20% was observed (Fig. 2). Until the 15% PEG treatments, the leaves showed no obvious rolling or color change. In the 20% PEG treatment, leaves started to roll slightly with leaf tip drying.

Figure 2. Effect of PEG concentration on TN rice seedling growth and root development after 7 days. (A) Representative images of rice seedling morphology and (B) Corresponding root development under 0%, 5%, 10%, 15%, and 20% PEG treatments. The scale bar represents 2 cm

Starting from 10% PEG, increasing PEG content lowered shoot length. The results showed a considerable decrease at 15% and 20% PEG treatments, with 20.8 ± 0.4 cm and 13.1 ± 0.4 cm, respectively compared to control (22.4 ± 1.0 cm) (Fig. 4A). Compared to the control (0.17 ± 0.007 g), fresh shoot weight decreases across treatments, with the greatest drop at 15% (0.14 ± 0.013 g) and 20% PEG (0.12 ± 0.002 g) (Fig. 4D). Root length increased at 5% PEG (3.4 ± 0.07 cm) and was maintained across different PEG treatments (Fig. 4B). Root numbers remain constant at 0% and 5% (7.9 ± 0.4 cm and 7.6 ± 0.4 cm) but decreases at higher concentrations (Fig. 4C). Root fresh biomass (Fig. 4G) fluctuated slightly but did not differ across treatments. The steady increase in dry root biomass (Fig. 4H) and decrease in water content (Fig. 4I) from the control (88.6 ± 0.6%) to 20% (85.5 ± 0.4%) were also observed.

Figure 4. Effects of PEG concentration on TN rice cultivar. (A) shoot length; (B) root length; (C) number of roots; (D) shoot fresh weight; (E) shoot dry weight; (F) shoot water content; (G) root fresh weight; (H) root dry weight; (I) root water content; (J) total chlorophyll and carotenoid; (K) proline content; (L) total proteins; (M) guaiacol peroxidase (GPX) activity; (N) ascorbate peroxidase (APX) activity; (O) superoxide dismutase (SOD) activity. Data are presented as the mean ± SE

TN rice seedling could cope with high salt treatment

The 120 mM NaCl treatment significantly impacted plant survival in Nipponbare (Fig. 1B). Plant height dropped, and leaves were dried. In addition, the leaves of Nipponbare grew softer in the presence of NaCl. Some leaves withered and fell apart. Some seedlings died within the 7-day of salt stress treatment salt stress. On the other hand, all TN seedlings survived after 7 days of salt treatments despite the growth was gradually affected by the increase of NaCl concentration in the solution (Fig. 3). The seedlings coped well with 30 mM NaCl treatment as no obvious effect on physiology has been observed. At 60 and 90 mM NaCl, the TN seedling could maintain shoot growth with only slight stress symptoms including curling and turning yellow at the tips. At 120 mM NaCl, shoot elongation was severely slowed down, leading in shorter lengths with increased rolling and yellowing. Despite these severe effects at the highest salt treatment, all TN seedling remained alive.

Figure 3. Effect of salt concentration on TN rice seedling growth and root development after 7 days. (A) Representative images of rice seedling morphology and (B) Corresponding root development under 0 mM, 30 mM, 60 mM, 90 mM, and 120 mM NaCl concentration. The scale bar represents 2 cm

Detailed analyses of TN seedlings showed that shoot length was not affected by 30 mM NaCl treatment but decreased gradually when the NaCl concentration increased from 30 to 120 mM NaCl (20.79 ± 0.42 cm to 8.68 ± 0.31 cm) (Fig. 5A). Shoot fresh weight, dry weight and water content also followed the same tendency (Fig. 5D, E, F). The root length was not affected by 30 mM NaCl treatment but declined gradually when the NaCl concentration increased from 30 to 90 mM (2.81 ± 0.17 cm). Increasing to 120 mM NaCl did not reduce further the root length (Fig. 5B). The root number (Fig. 5C), however, response to salt stress in a different pattern. The lowest root number was observed in 60 and 90 mM NaCl treatments. At 120 mM NaCl, the root number was restored. The effect of salt stress on the root biomass of the TN seedling was much more complex with no clear tendency. The 90 mM NaCl treatment showed the strongest negative effect on both root elongation and root water content, leading to a significant reduction of total root fresh weight (Fig. 5G).

Figure 5. Effects of NaCl concentration on TN rice cultivar. (A) shoot length; (B) root length; (C) number of roots; (D) shoot fresh weight; (E) shoot dry weight; (F) shoot water content; (G) root fresh weight; (H) root dry weight; (I) root water content; (J) total chlorophyll and carotenoid; (K) proline content; (L) total proteins; (M) guaiacol peroxidase (GPX) activity; (N) ascorbate peroxidase (APX) activity; (O) superoxide dismutase (SOD) activity. Data are presented as mean ± SE

Effects of drought and salt stress on chlorophyll, carotenoid and proline content of TN seedlings

In this study, different PEG concentrations showed no effect on the total protein of the TN seedlings (Fig. 4L). However, the increase of PEG in the solution led to the rise of chlorophyll, carotenoid and proline levels. The total chlorophyll and carotenoid were stable in 5% PEG treatment but started to rise when the PEG concentration increased further (Fig. 4J). While the total carotenoid raised gradually with the increasing of PEG throughout the experiment, the total chlorophyll content reached the highest value at 15% PEG (0.19 ± 0.04 µg/g) and remained unchanged when PEG concentration was increased to 20% (0.19 ± 0.007 µg/g) (Fig. 4J). The total chlorophyll in high PEG treatments was almost double the value obtained from the control condition. Proline content increased considerably, doubling from 0% to 5% (1.87 ± 1.24 µM/g), stabilized at 5% to 15%, and reached the highest value at 20% PEG (4.87 ± 3.04 µM/g) (Fig. 4K).

Under salt stress, the biochemical activities of the TN seedlings showed a different pattern. Protein content gradually rose with increasing NaCl concentrations, from 3.091 ± 0.120 mg/g in the control to 5.575 ± 1.271 mg/g in the 120 mM NaCl treatment with a slight drop at 90 mM NaCl (3.861 ± 0.190 mg/g) (Fig. 5L). The total chlorophyll level peaked at 30 mM NaCl (0.945 ± 0.041 µg/g) and then decreased gradually with the increase of the NaCl (Fig. 5J). The total carotenoid content remained stable when the NaCl content in the solution increased to 60 mM. However, when the NaCl content in the solution went beyond 60 mM, we observed a significant drop in the total carotenoid content to 0.455 ± 0.039 and 0.416 ± 0.041 µg/g in 90 and 120 mM NaCl treatments, respectively (Fig. 5J). Different from chlorophyll and carotenoid, the proline content increased with the increase of NaCl in the solution, with the highest values observed at 90 and 120 mM NaCl treatments, approximately sevenfold higher than the proline content in plants grown in control conditions (Fig. 5K).

Antioxidant enzyme activities change changed differently under drought and salt stress in TN rice cultivars

In terms of enzyme activity under drought stress (Fig. 4N), APX activity increased with PEG concentration and reached a particularly high value at 20% PEG (11.77 ± 0.95 UI/mg), six folds higher than in the control condition. GPX activity (Fig. 4M) fluctuated with the change of PEG in the solution, with the lowest activity observed at 15% PEG (0.665 UI/mg). SOD activity remained unchanged across the PEG treatments (Fig. 4O).

Under salt stress, the activity of SOD (Fig. 5O) and APX (Fig. 5N) increased from control to 120 mM NaCl treatment (from a range of 0.085 ± 0.011 to 0.140 ± 0.034 UI/mg for SOD followed by 0.996 ± 0.128 to 2.328 ± 0.311 UI/mg for APX). GPX activity showed a sharp decrease in all salt treatment and fluctuated at low level with the change of salt concentration in the medium (Fig. 5M).

Comparison of SSR markers between the TN and Nipponbare cultivars

All four SSR markers associated with drought tolerance were detected in the genome of both Nipponbare (sensitive) and TN (survey) cultivars, although the size of the PCR products was different between them (Fig. 6A). Three markers—RM164, RM223, and RM228—generated smaller PCR products for TN cultivar compared to Nipponbare. In the case of RM263, a larger size of PCR product was observed for the TN cultivar. Among four Saltol SSR markers, three—RM493, RM562, and RM3412—were detected in both varieties and allowed to distinguish them based on the significant difference in the size of their respective PCR products (Fig. 6B). Compared to Nipponbare, the two markers RM562 and RM3412 generated smaller PCR products for TN, while the marker RM493 gave a larger PCR product for this surveyed cultivar. The size differences from the electrophoresis results were consistent with those verified by Illumina sequencing data (Table 1). In our study, we were not able to detect any PCR product for the RM23 marker in TN cultivars.

Figure 6. Agarose gel electrophoresis of SSR markers amplified from Nipponbare and TN rice cultivars. (A) Markers related to drought stress. (B) Markers related to salt stress. The negative control contained no DNA templates

Table 1 . SSR region variants detected through Illumina sequencing data.

SSR markers	Expected PCR product size (Nip/TN) (bp)	Variants of Tai Nguyen	Stress
RM223	148/138	10 nucleotide deletion in the repeat CT region	Drought
RM228	114/108	6 nucleotide deletion in the repeat AG region
RM263	186/215	26 and 3 nucleotide insertions in the repeat CT and repeat T regions
RM164	265/253	12 nucleotide deletion in the repeat TG region
RM23	136/145	6 nucleotide deletion (GTGCGC), 8 nucleotide deletion in repeat TG regions, 20 nucleotide insertion in repeat AG regions, and 3 nucleotide insertion in repeat TAC	Salt
RM493	211/247	36 nucleotide insertion in the repeat GAA region
RM562	245/233	12 nucleotide deletion in the repeat GAA region
RM3412	211/201	10 nucleotide deletion in the repeat GA region

To investigate the plant responses to drought stress, PEG with high molecular weight is often used to manipulate the osmotic potential of the growth solution (Hellal et al. 2018; Susilawati et al. 2022). For the Nipponbare cultivar, PEG concentration above 15% (equivalent to -250 KPa for PEG 6000) was reported to completely inhibit plant growth and even be lethal (Lu et al. 2009; Michel and Kaufmann 1973). Indeed, in our study, most of the Nipponbare seedlings died after 7 days exposed to 20% PEG solution (-500 KPa). The ability to survive in such a high drought level of the TN seedling suggested the potential drought tolerance of this cultivar. The delay of leaf rolling is also a parameter suggesting the drought tolerance (Kim et al. 2020; Wopereis et al. 1996). The study on two drought sensitive rice cultivars, IR20 and IR72, showed that soil water potential lower than -370 KPa led to leaf rolling and dead in young seedlings, and the ratio of dead leaves reached 35-40% when the soil water potential dropped to -500 KPa (Wopereis et al. 1996). In this study, at 20% PEG (~ -500 KPa) only started to trigger the rolling of leaf tips of the TN seedlings, and no completely dead leaf has been observed after 7 days of exposure to this drought treatment, suggesting a better drought tolerance of this cultivar compared to the other two cultivars previously mentioned. A study on seminal root elongation of the Nipponbare cultivar in response to different PEG concentration indicated that root growth was strongly induced by low PEG concentration (5% - 10%) and was almost completely suppressed when the PEG concentration is higher than 12% (Susilawati et al. 2022). We also observed the same tendency where the root length of TN seedlings significantly increased in 5% PEG treatment and gradually decreased with further increase of PEG concentration. However, unlike the Nipponbare seedlings, the TN seedlings were able to maintain a low level of root growth at 15% and 20% PEG treatments as well as increase the total root dry weight. According to Kim et al. (2020), drought-tolerant rice varieties could access deeper soil water sources thanks to their deeper root systems and density (Kim et al. 2020). As root growth involves cell division and expansion, both require energy from photosynthesis part and turgor pressure, our results suggested that the TN seedlings were able to retain an effective root turgor pressure required for root growth while maintaining a basal level of photosynthesis to fuel the process. Indeed, we observed a rather stable water content in root as well as the accumulation of carotenoids and chlorophyll in the shoot of the TN seedlings. These observations also suggested that the TN cultivar can efficiently adjust the water use upon sensing drought stress to increase the survivability. Melandri et al. (2021) reported that upland and aerobic rice cultivars lose less water than the lowland IR64 cultivar, a popular commercial cultivar in Vietnam (Melandri et al. 2021). In our study, despite being a lowland cultivar, the TN seedlings were able to maintain a rather stable root and shoot water content at very high drought treatments.

Our biochemical analyses showed that the TN cultivar has drought adaptive properties of a drought-tolerant cultivar as it increased the total chlorophyll and carotenoid to protect against oxidative damage by stabilizing photosynthetic systems and decreasing ROS damage (Uarrota et al. 2018; Wang et al. 2022). There are multiple evidence showing that the ability of accumulation of chlorophyll and carotenoids during drought stress is linked to drought-tolerance in different plant species (Çiçek et al. 2015; Nahakpam 2018; Syamsia et al. 2018; Talbi et al. 2020; Yan et al. 2024; Yang et al. 2023). In these studies, upon drought stress, the change in carotenoids and chlorophyll were not uniformed between cultivars. Instead, drought-tolerant cultivars were able to either maintain or accumulate these compounds while sensitive cultivar showed a clear decrease of both carotenoids and chlorophyll. During abiotic stress, the accumulation of proline helps plant to cope with extreme conditions by lowering the osmotic potential within the cytoplasm, stabilizing proteins and membranes, scavenging free radicals, and maintaining cellular homeostasis (Dien et al. 2019; Gharsallah et al. 2016). Our observation of significant increase in proline content in 20% PEG but not before indicated that this cultivar can cope very well with water potential above - 250 KPa.

Salt stress affects multiple biological processes, inhibiting plant growth (Dzinyela et al. 2023). In our study, increased NaCl concentrations shortened shoots and roots and reduced shoot fresh weight, dry weight, and water content, which is consistent with many prior studies on the negative impacts of salt stress on plant growth (Dikobe et al. 2021; Vázquez-Glaría et al. 2021). Shoot water content declines as greater salt concentrations lower osmotic potential, limiting root water absorption (Lu and Fricke 2023). Ion balance, as well as water status, important for normal physiological along with biochemical functions, are disrupted by salt stress (Li et al. 2024). Compromised water and nutrient absorption stunts plant growth, negatively affects root development, as well as reduces plant biomass.

Total protein fluctuated more in salt-tolerant rice, according to research of Das (Das et al. 2019). Under salt stress, mitochondrial antioxidant defense proteins are upregulated (Athar et al. 2022). Salinity forces plants to adapt by modifying the Krebs cycle, metabolism, and membrane transport system, while also enhancing antioxidant defense proteins to maintain cellular homeostasis (Athar et al. 2022). The ability of the TN cultivar to maintain protein stability under drought and upregulate protective proteins under salt stress suggested its stress tolerance (Abdallah et al. 2016; Hu et al. 2023). In this study, cultivar salt stress, we observed both chlorophyll and carotenoid decrease when the concentration of NaCl in the solution excess 60 mM cultivar. Xanthophylls and carotenes, the two classes of carotenoids (Thomas and Johnson 2018), are important for plant antioxidant defense and are easy to be destroyed by oxidative reaction (Munné-Bosch and Alegre 2000). This phenomenon explains the decrease of the total carotenoids under high salt stress conditions observed in our study, as commonly reported in other plant species (Farooq et al. 2019; Khalil 2020; Stępień and Kłbus 2006). The proline content was found to rise rapidly in plants when exposed to higher salt concentrations (Gharsallah et al. 2016). Study on O. australiensis JC 2304 and Pokkali, two salt tolerant rice cultivars, found that the proline content increase after two days exposed to salt stress (Choudhary et al. 2009; Nguyen et al. 2021). In our study, this adaptive response was also observed in the TN cultivar.

Dehydration and salinity disrupt the electron transfer chain in photosynthesis, leading to the over reduction of electron carriers, enhancing the formation of ROS (Cruz de Carvalho 2008). ROS are signal molecules for cellular homeostasis, but overexposure can damage cell components, inhibit enzyme activities, and cause programmed cell death. Plant cells protect themselves from ROS with SOD, CAT (catalase), APX, and GPX enzymes. SOD is the first line of defense against oxidative stress, and drought stress enhances it in rice (Panda et al. 2021). SOD activity maintains redox balance and protects biological components from oxidative damage under stress (Alscher et al. 2002). APX uses ascorbate to donates electrons to transform H₂O₂ into water (Sofo et al. 2015) while GPX reduces H₂O₂ with phenolic compounds such as guaiacol (Van Doorn and Ketsa 2014). In our study, SOD activity increased with salt content in rice, consistent with findings from salinity studies on Cicer arietinum, Beta vulgaris, A. tricolor, and Brassica juncea (Che et al. 2020; Sarker and Oba 2020). Rising APX activity during drought stress aids antioxidant defense, reducing H₂O₂ and safeguarding cells from oxidative damage (Sofo et al. 2015; Zhang et al. 2013). Our investigation showed that under salt stress, TN cultivar APX activity rises with salt level, similar to what has been reported for the White ponni and BPT-5204 cultivars (Thamodharan and Pillai 2014). The GPX activity during salt stress is, however, more complicated. The GPX activity is affected by multiple factors including genetics, treatments, and interactions (Rohman et al. 2024). That is why upon salt stress, GPX activity was reported to decrease in some species while increase in others (Monsur et al. 2020; Rabiei et al. 2020; Rohman et al. 2024). In the case of TN cultivar, we observed a significant drop of GPX activity upon salt stress, suggesting the antioxidant capacity during salt stress of this cultivar mainly relied on SOD and APX.

We observed differences between Nipponbare and TN cultivars in the PCR product size of all tested SSR markers in our study. Among the tested drought tolerant-related SSR markers, RM164 is linked to the release of momilactones A and B, which help it tolerate drought and salinity (Xuan et al. 2016), while RM263 and RM228 affect rice drought resistance by leaf rolling, root water uptake, and the root/shoot ratio which is important adaptive trait of rice under drought stress (Kim et al. 2020; Michael Gomez et al. 2010; Nguyen et al. 2008; O’Toole and Cruz 1980; Wang et al. 2023; Wening et al. 2021). RM228 is also linked to flavonoids, which are antioxidants in plant cells (Shao et al. 2011). RM223 was shown to have tight connection with the dry weight of seedlings (Sanghamitra et al. 2021). In practice, the three markers RM164, RM223, and RM228 were proven to be efficient in identifying drought-tolerant cultivars in Egypt (Alafari et al. 2024; Gaballah et al. 2021; Kumar et al. 2005). In Vietnam, RM263 allowed efficiently identifying Vietnamese drought-tolerant rice cultivars (Nguyen et al. 2008).

The Saltol markers are however much less understood as about 25% of genes in the saltol region have not been completely described (Waziri et al. 2016). However, the SSR markers within this region have been successfully used in different rice salt tolerance studies in Vietnam, Thailand, and India (Huong et al. 2020; Kordrostami et al. 2016; Liu et al. 2023). In this study, the three saltol SSR markers giving PCR products are located in the upstream regions, either promoter or 5’-UTR, of genes in the rice chromosome 1. The gene linked to RM493 encodes a protein of unknown function. RM3412 locates in the promoter region of the two genes Os01g20770 and Os01g20780. Although both genes have not been characterized, the Os01g20770 gene encodes a protein belonging to the plant pentatricopeptide family responsible to regulate gene expression in plant organelles including mitochondria and chloroplasts (Barkan and Small 2014), suggesting its connection with stress response. Interestingly, the SSR marker RM562 locates in the 5’-UTR region of OsNox1/OsRbohB encoding a plasma membrane NADPH oxidase, responsible to produce ROS in plant during drought stress (Shi et al. 2020; Wang et al. 2013). The difference in PCR product size between two cultivars is evidence of change in the genome sequence of genes relating to drought and salt tolerance, suggesting potential change in the mechanism and intensity of stress responses of the TN cultivar. By analyzing the precise insertion and deletion via comparing the available genomes of the two cultivars, our study could facilitate further functional analyses of genes and their regulation via the promoter sequence which will, in turn, validate the direct link of SSR alleles with stress tolerance in rice. In the Mekong Delta, as drought stress is often accompanied by salt stress due to saltwater intrusion during dry season, it is essential to identify rice cultivar which can tolerate both drought and salt stress. In-depth study on stress tolerance-related markers will help to speed up this selection process.

This research is funded by University of Science, VNU-HCM under grant number T2024-38.

T-HN. and VAL. designed the project and revised the manuscript. All authors prepared the manuscript. T-PD, VAL, M-TT, and P-KN conducted the experiments and analyzed the data. MYH supported collecting the results of physiology experiments. VAL performed NGS data analysis.