Skip to main content
NCBI home page
Scientific Reports logoLink to Scientific Reports
. 2026 Jan 16;16:4693. doi: 10.1038/s41598-025-34961-w

Enhancing drought stress tolerance of NIL-SUB1DRO1 rice strain through different seed priming treatments

Alex Tamu 1,2, Aquilino Lado Legge Wani 1,4, Ibrahim Soe 1,2, Emmanuel Odama 5, Taiichiro Ookawa 6, Abdelbagi M Ismail 7, Jui-Ichi Sakagami 1,3,✉
PMCID: PMC12868621  PMID: 41545402

Abstract

Drought stress is a major abiotic factor that significantly reduces rice yield and productivity. This study analyzed the effectiveness of various seed priming techniques in enhancing drought stress tolerance in NIL-SUB1DRO line with an IR64 genetic background, known for its combined tolerance to flooding and drought stress. A complete randomized factorial design with five replications was employed, testing osmo-priming, halo priming, hydro priming, and non-primed control under drought and well-watered conditions. Drought stress markedly reduced plant height, shoot, and root biomass, leaf area, photosynthetic activities, and water relations traits (relative water content and leaf water potential) in non-primed seedlings, whereas seed priming treatments, especially osmo-priming and halo-priming treatments, significantly enhanced plant height, shoot, and root biomass, SPAD value, Fv/Fm, relative water content, leaf water potential, and photosynthetic rate in NIL-SUB1DRO1. Strong positive correlations between SPAD value, photosynthetic rate, Fv/Fm, biomass biomass-related traits. These findings suggest that seed osmo-priming is a promising strategy to enhance drought stress tolerance in NIL-SUB1DRO rice line.

Keywords: Chlorophyll fluorescence, Photosynthetic rate, Polyethylene glycol, Potassium nitrate, Seed priming

Subject terms: Physiology, Plant sciences

Introduction

Rice (Oryza sativa L.) is a major food crop and a primary source of calories for over half of the world’s population1. Rice is a C3 crop in the Poaceae family. It is diverse in adaptation, occupying larger production areas in the tropics, subtropics, semiarid tropics, and temperate regions of the world2. A stable and sufficient rice supply is, therefore, essential for food security, poverty reduction, and national economic development3 for major rice-producing countries. In rainfed areas, drought remains a leading abiotic stress affecting rice production, as it directly affects plant growth, development and economic yield4. It triggers a series of physio-morphological and molecular alterations5–7. Like several other abiotic stresses, drought induces the overproduction of reactive oxygen species (ROS) like superoxide radicals, singlet oxygen, hydrogen peroxide, and hydroxyl radicals8,9. When protective mechanisms are compromised, ROS damages vital structural and functional components of plant systems9.

Strategies to combat drought stress (DS) include breeding drought-tolerant cultivars, application of growth regulators, osmo-protectants, mineral nutrients, and modifying cropping patterns to escape stress during critical stages10. Breeding drought-tolerant cultivars is time-consuming and technically demanding due to the complex genetic architecture of DS, the involvement of multiple stress factors, and limited knowledge of drought-responsive genes11. Roots are the main components of plant adaptation under drought conditions. Plants with drought avoidance are characterized by deep roots and abundant coarse roots with high branching and penetration abilities12. This root architecture may enhance water acquisition when sufficient moisture is available in deeper soil layers. Drought-tolerant rice varieties typically possess smaller, denser root systems that enhance water uptake from deeper soil layers, thereby contributing to drought avoidance13. Three major QTLs have been identified that are associated with steep root growth angles14, among which DEEPER ROOTING1 (DRO1) remains the most prominent. Genotypes, like NIL-SUB1DRO1 contain SUB1 and DRO115, combining tolerance to submergence and deeper rooting for drought avoidance. However, the development and dissemination of such genetically improved lines require time, technical expertise, and substantial investment. Thus, finding low-risk, low-cost, and practical methods to mitigate DS is imperative. Seed priming is a simple, cost-effective, and considerable approach for promoting faster seed germination, early seedling growth, and improving yield under control and stress conditions16. Priming is a controlled hydration–dehydration treatment that initiates pre-germinative metabolic activity without allowing radical emergence17 and is known to enhance plants’ tolerance to abiotic stresses. It also facilitates the accumulation of osmolytes (proline, glycine-betaine, polyamines) through altered metabolic processes18. NIL-SUB1DRO1 was obtained by crossing NIL-SUB1(a cross between IR64 and the submergence tolerant FR13A) with NIL-DRO1 (a cross between drought susceptible IR64 and drought resistance rice Kinandang Patong)15. An indica cultivar, FR13A, exhibits - high tolerance to flooding and possesses the capability to endure complete submergence for two weeks due to a significant quantitative trait locus (QTL) designated as Sub1, located near the centromere of chromosome 9. Kinandang Patong contains DRO1, a major QTL associated with the deep rooting of rice in upland field conditions. This QTL regulates the architecture of the root system and enhances rice yield under drought conditions15.

Although NIL-SUB1DRO1 lines with an IR64 genetic background already exhibited tolerance to flooding and drought stress (DS), the potential of seed priming to further strengthen their drought resilience has not been fully investigated. Therefore, this study evaluated the effect of various seed priming methods in enhancing drought tolerance in the NIL-SUB1DRO1 genotype at the seedling stage under DS conditions. The findings provide insights into the drought response of NIL-SUB1DRO1 and its potential for further improving its tolerance resilience to drought.

Methodology

Site and materials

The experiment was conducted in a greenhouse at the Graduate School of Agriculture Science, Kagoshima University, Japan. The relative humidity and air temperature in the greenhouse was measured with sensor equipped with a data logger (RTR-503, T&R Cooperation, Japan) during the experimental period. The average temperature and humidity in the greenhouse were 23.5℃ and 52%, respectively. The experiment comprised NIL-SUB1DRO1 (submergence and drought-resistant) line obtained from the Tokyo University of Agriculture and Technology, Japan, in collaboration with Kagoshima University, Japan. Figure 1 presents detailed meteorological data during the DS period.

Fig. 1.

Fig. 1

Open in a new tab

Changes in experimental air temperature and relative humidity during the Drought stress (DS) and well-watered period.

Seed priming

The seeds were soaked in salt water with a specific gravity of 1.13. They were washed and rinsed with distilled water and later air-dried to 13.6% moisture content using a grain moisture tester (Riceter, Kett Electric Laboratory, Tokyo, Japan). Different priming treatments were achieved by placing the seeds into 100 ml of distilled water (hydro-priming), 2 g KNO3, (halo-priming), and 5 g polyethylene glycol (osmo-priming) in a solution ratio of 1:3 [W(g): V(ml)] at 28℃ in a natural convection incubator (SIB35, Sanyo, Tokyo, Japan) for 24 h. The seeds were then removed from the solution, rinsed twice with distilled water, and air-dried at 28℃ until the initial seed weight was obtained. Non-primed seeds served as the control treatment.

Experimental design

A complete randomized factorial design with five replications was employed, testing osmo-priming, halo-priming, hydro-priming, and non-primed control under drought and well-watered conditions. Selected seeds were placed in Petri dish lined with filter paper moistened with distilled water and incubated at 28 °C for 48 h to allow germination. After germination, primed and none-primed seeds were directly sown into polyvinyl chloride pipes (8 cm inner diameter × 40 cm height), each filled with 2.6 kg of commercial soil supplemented with 10 g of N.P.K (8.8.8) compound fertilizer. For each treatment, three pregerminated seeds were sown per pipe and later thinned to 1 seedling per pipe. The seedlings were irrigated daily for 21 days, after which DS was induced by withholding irrigation for 22 days. In all treatments, soil moisture sensors equipped with a data logger (Em50 Series; METER Group, Inc. USA) were fitted at a depth of 10 cm to determine soil moisture content in the pipes. Soil moisture data were recorded within 60-min intervals between measurements throughout the experiment using a ZL6 Basic Datalogger (Meter Group, Inc. Pullman, WA, USA).

Data collection

Growth performance

Shoot parameters including plant height, leaf area, leaf fresh weight, and soil plant analysis development (SPAD) values were measured before drought treatment and during DS. Plant height was measured from the stem’s base to the flag leaf tip. Three shoot samples per variety and treatment were collected, and the leaves were separated for leaf area measurement using a digital image analysis system (LIA32, Nagoya University, Nagoya, Japan). Harvested leaves and stems were used to determine -leaf and shoot dry weights (DW) after oven dried at 80℃ for 48-h.

SPAD values

The mean SPAD values of primed and non-primed seedlings per genotype and treatment were determined at the tip, middle, and base of the third fully expanded leaf, and averaged as the SPAD reading for leaf chlorophyll index, using an SPAD-502 leaf chlorophyll meter (Konica Minolta Japan Inc., Tokyo, Japan).

Chlorophyll fluorescence (Fv/Fm)

The PSI AquaPen-P AP-P 100 instrument (Photon Systems instruments, Drasov, Czech Republic) was clipped onto the fully expanded third leaves of each plant after 2 h of adaptation to the dark. Quantum yield was measured as the efficiency of photosystem II, which corresponded to the Fv/Fm in dark-adapted leaves.

Leaf gas exchange parameters

Leaf gas exchange parameters including photosynthesis rate (A) and transpiration rate (E) were measured on the central leaflet of fully expanded flag leaf under control and drought stress. The measurements were taken using a portable gas exchange system (LI 6400XT, LI-COR Inc., Lincoln, NE, USA) equipped with a standard 2 × 3 cm clear chamber (6400-08). The block temperature was maintained at 28 °C with vapor pressure deficit regulated at an air flow rate of 500µmol s−1. During measurements, leaves were acclimated under saturating light (1500 µmol m−2 s−1 and a CO2 concentration of 400µmol mol−1 inside the chamber.

Relative water content

The second fully expanded young leaf of two randomly selected plants from each genotype within treatments was used to measure relative water contents. Freshly harvested leaves were cut 5 cm from the lamina base, weighed, and placed in test tubes submerged in distilled water for 4 h at 28 °C. After incubation, the leaves were blotted dry, and the turgid weight was recorded using a precision scale. Leaf samples were then oven-dried at 80 °C for 24 h and weighted (DW). The leaf relative water content was calculated using this formula:

graphic file with name d33e435.gif

where FW = fresh weight of the sample immediately after collection.

DW = dry weight of the sample after oven-dried at 80 °C for 24 h.

TW = turgid weight of the sample after hydration for 4 h and blotted dry.

Statistical analysis

Data was analyzed using the IBM SPSS software package (version 27.0.1.0). Differences in mean values among priming treatments under DS and control conditions were assessed using two-way analysis of variance (ANOVA), followed by Tukey’s HSD test at a significant level p < 0.05%. Pearson’s correlation analysis was conducted to examine potential associations among the measured traits.

Results

Experimental soil conditions

This figure illustrates soil moisture dynamics under drought and well-watered conditions across different priming treatments (Osmo priming, halopriming, and hydropriming) and their controls. Under WW, soil moisture remained stable around (0.18–0.19 m3m−3) throughout the 22 days period. No differences were observed among the control treatment, indicating that soil moisture was uniformly sustained. Under DS, soil moisture declined progressively from (~ 0.18 m3m−3 on day 1 –0.03 m3m−3 by day 22). This suggests that the reduction reflects evapotranspiration driven by water loss due to imposed drought stress (Fig. 2).

Fig. 2.

Fig. 2

Open in a new tab

Changes in the experimental soil volumetric moisture content under drought and well-watered across seed priming treatments of NIL-SUB1DRO1. OS + D; Osmo-priming (Drought), HA + D; Halo priming (Drought), HY + D; Hydro priming (Drought), OS + C; Osmo priming (Control), HA + C; Halo priming (Control), and HY + C; Hydro priming (Control).

Effect of priming on plant growth

Significant (p < 0.05) differences among priming treatments of NIL-SUB1DRO1 were observed in plant height, leaf area, root and shoot dry weight (DW) under both water regimes (Fig. 3). Under well-watered conditions, all the seed priming enhanced plant height with Osmo priming recorded the tallest plant height by (18.5% higher than the non-primed of NILSUB1DRO1), while all the priming techniques significantly (p < 0.05) enhanced the leaf area, with osmo-priming recorded the largest leaf area (Fig. 3b). Root and shoot dry weight were also improved osmo-priming recorded the largest root and shoot dry weight by 6% and 7% respectively, compared to other priming treatments, with least root and shoot recorded in non-primed controls. Drought stress (DS), impaired seedling growth and development of NIL-SUB1DRO1 non primed treatments. However, seed priming significantly mitigated the DS effects on NIL-SUB1DRO1 primed seedlings. Under DS, osmo primed seedlings exhibited 19.5% greater plant height, 26% leaf area, and maintained 22% and 19% root and shoot DW, respectively, compared with non-primed seedlings. Halo and hydro priming also conferred significant improvements, thought to a less extent (Fig. 3a, b, c and d). Overall, osmo priming consistently enhanced growth and biomass traits under both drought and well-watered, indicating it effectiveness in promoting seed vigor and drought tolerance.

Fig. 3.

Fig. 3

Open in a new tab

Effect of seed priming on NIL-SUB1DRO1 growth s under well-watered (WW) and drought stress (DS) treatments. Plant height (a), LA; leaf area (b), RDW; Root dry weight (c), and SDW; shoot dry weight (d). Treatments include Control (non-primed), halo primed, hydro primed and Osmo primed. (n = 5). Different lowercase letters indicate significant differences among treatments at p < 0.05. Error bars represent standard deviations.

Effect of priming on gas exchange

Figure 4 illustrates the effect of different seed priming techniques on gas exchange and water relation parameters of NIL-SUB1DRO1 seedlings under well-watered and drought stress conditions. Under well-watered conditions, all the different seed priming treatments significantly (p < 0.05) improved relative water content, leaf water potential (Ψleaf), photosynthetic rate, SPAD value, Fv/Fm, and transpiration rate compared to no priming treatments, with Osmo priming treatments consistently performed better than other seed priming treatments (Fig. 4). DS resulted in a significant decline in all the measured traits, yet all the different seed priming maintained higher water relation and photosynthetic traits than the non-primed. However, Osmo priming seedlings exhibited significantly higher RWC 8%, Leaf water potential 30%, photosynthetic rate 24%, SPAD value 9% compared with non-primed seedlings (Fig. 4). Halo priming led to a 5% improvement in relative water content and a 20% enhancement in the photosynthetic rate compared with the control. Across both water regimes, osmo priming was the most consistent thereby mitigating drought-reduce reductions in photosynthetic efficiency and maintaining higher chlorophyll content (SPAD), water status, and quantum yield (Fv/Fm) compared to non-priming treatment (Fig. 4).

Fig. 4.

Fig. 4

Open in a new tab

Effect of seed priming treatments on the gas exchange of NIL-SUB1DRO1 under WW and DS. Relative water content (a), leaf water potential (b), photosynthetic rate (c), transpiration rate (d), SPAD value (e), and Fv/Fm (f). Treatments include Control (non-primed), halo primed, hydro primed and Osmo primed. (n = 5). Different lowercase letters indicate significant differences among treatments at p < 0.05. Error bars represent standard deviations.

Correlation and principal component analysis (PCA)

Pearson correlation analysis was performed to evaluate the relationships among all measured traits and to explore the effects of different priming agents on physiological and photosynthetic parameters (Fig. 6). A strong positive correlation was observed among SPAD, photosynthetic rate, quantum yield (Fv/Fm), and biomass-related traits (root and shoot DW). These findings suggest that plants with higher chlorophyll content and photosynthetic efficiency exhibit greater carbon fixation and biomass accumulation under drought conditions (Fig. 6a, b, c, d, and e). A positive linear correlation was observed between leaf water potential and photosynthetic rate, indicating that increases in relative water content are associated with higher net photosynthetic rates. PCA was performed to assess the contributions of various physiological and photosynthetic indices to the total variability in NIL-SUB1DRO1 responses to seed priming and DS (Fig. 5). Two principal components had eigenvalues exceeding 1, with the biplot depicting PC1 and PC2 accounting for 92.4% of the total variance in the data (Fig. 5). This revealed distinct patterns among treatments for morphophysiological and photosynthetic traits. PC1 accounted for 82.9% of the variation, indicating that a substantial proportion of the differences among samples could be explained by this single component. PC2 explained 9.5% of the variance, suggesting that the two principal components effectively explained the major patterns in the data. The dominance of PC1 suggests that the primary factor differentiating the samples is strongly related to the combined influence of root DW, photosynthetic rate, and Fv/Fm, along with the opposing influence of transpiration. Root DW, photosynthetic rate, and Fv/Fm were positively correlated, indicating that treatments that increase root biomass also led to higher photosynthetic rate and quantum yield. The location of the clusters in the PCA space enables the characterization of each group based on its dominant variables. Cluster one was characterized by high transpiration rates and potentially lower root DW, photosynthetic rate, and Fv/Fm. Cluster two showed higher root DW, photosynthetic rate, and Fv/Fm. Clusters three and four represented other distinct combinations of these physiological traits.

Fig. 6.

Fig. 6

Open in a new tab

Relationships between photosynthetic rate and SPAD (a), leaf area (b), Root DW; root dry weight (c), Shoot DW; shoot dry weight (d), Fv/Fm (e), and RWC; relative water content of NIL-SUB1DRO1 seed priming under well-watered and DS. Asterisks indicate the level of correlation significance: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Fig. 5

Open in a new tab

PCA illustrates variance and trait patterns via biplot of photosynthetic rate, Fv/Fm (quantum yield), root dry weight, and transpiration rate of NIL-SUB1DRO1 under well-watered and DS.

.

A scatter plot analysis

Figure 7. presents scatter plot analysis showing the relationship among physiological and photosynthetic traits under well-watered and drought stress conditions. Significant positive relationships were observed for SPAD value (r = 0.68*), photosynthetic rate (r = 0.45*), and transpiration rate (E), (r = 0.57*) (Fig. 8a and f,8 h). Stronger positive linear relationships were detected for root dry weight (r = 0.84**), plant height (r = 0.70*), shoot dry weight (r = 0.95**), leaf area (r = 0.91**), and relative water content (r = 0.73*) (Fig. 7b-g).

Fig. 7.

Fig. 7

Open in a new tab

A scatter plot showing a relationship among different traits (a) SPAD, (b) Root dry weight, (c) Plant height, (d), Shoot dry weight, (e) Leaf area, (f) Photosynthetic rate (A), (g) Relative water content, and (h) Transpiration rate(E) under Well watered and Drought stress. Asterisks show the significance of each correlation: *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

DS leads to net water loss through transpiration due to restricted water uptake due to reduced soil moisture availability19. Seed priming hydrates the seed to activate pre-germination metabolic and biochemical processes before radical protrusion during phase II of seed germination20. This enhances the accumulation of osmolytes (proline, glycine-betaine, and polyamines) through altered metabolic processes18. An indica cultivar, FR13A, exhibits - high tolerance to flooding and possesses the capability to endure complete submergence for two weeks due to a significant quantitative trait locus (QTL) designated as Sub1, located near the centromere of chromosome 9. Kinandang Patong contains DRO1, a major QTL associated with the deep rooting of rice in upland field conditions. This QTL regulates the architecture of the root system and enhances rice yield under drought conditions14.

This study hypothesized that seed priming can further enhance drought stress tolerance of the NIL-SUB1DRO1 line with the IR64 genetic background, known for its tolerance to both flooding and DS. Therefore, this study evaluated the effect of various seed priming methods in enhancing drought tolerance of the NIL-SUB1DRO1 line at seedling stage under DS conditions. In this study, DS reduced the plant height of non-primed seedlings compared to primed seedlings (Fig. 3a). This is because the non-primed seeds lack pre-activation of stress response pathways, making seedlings more vulnerable to reduce photosynthesis and growth6 However, it was evident that the different seed priming methods significantly improved the plant height of NIL-SUB1DRO1 under DS. Several studies have shown that PEG osmo priming can improve growth, plant height, and leaf area in Medicago sativa L. seedlings21. The increase might have resulted from the influence of priming treatments on cellular expansion and elongation processes, potentially by modulating hormone-signaling pathways22. These findings are consistent with those reported before23where priming was found to increase the metabolic activities of seeds, resulting in a substantial increase in shoot length.

DS reduced leaf area of non-primed seedlings compared to primed seedlings. However, the extent of reduction was smaller in the osmo-primed and halopriming seedlings compared to the non-primed control. Notably, osmo priming mitigated the adverse effects of drought stress on this trait. This aligned with the findings of24. who reported that seed priming significantly improved leaf area in both rice types under drought and well-watered conditions, but osmopriming was more effective than hydropriming. This implies that seed priming enhances chlorophyll biosynthesis, stomatal regulation, and CO₂ assimilation efficiency, resulting in better energy supply for leaf growth and larger leaf area6. DS-impaired the growth of NIL-SUB1DRO1 non-primed seedlings, which resulted in low shoot and root biomass compared to primed seedlings. This could be as a result stomata closure, low chlorophyll content, and decreased CO2 assimilation6. Under DS, different priming treatments enhance both shoot and root biomass of NIL-SUB1DROL1, with performance more pronounced in osmo and halo primed treatments (Fig. 3c, d) The findings are also consistent with several reports on root dry mass in maize and sorghum, highlighting the beneficial effects of seed priming25.

In addition to chlorophyll content, photosynthetic efficiency, as indicated by the maximum quantum yield of photosystem II (Fv/Fm) has been widely used to assess rice tolerance to abiotic stresses26. In this study, non-primed seedlings showed substantial decline in Fv/Fm compared to the priming treatment under DS. However, Osmo-priming significantly enhanced the Fv/Fm of NIL-SUB1DRO1 under DS compared to non-primed treatment. (Fig. 4f), resulting in better growth of NIL-SUB1DRO1. According to27 primed seeds of rice and wheat, Saddiq et al.28 also showed an improvement in Fv/Fm under drought- and salinity-induced stress, respectively. Seed priming also boosted photosystem activity in rice seedlings by markedly reducing the extent of photoinhibition and improving photosynthetic efficiency29.

Chlorophyll concentration plays a critical role in determining the rate of photosynthesis. Under DS conditions, chlorophyll pigment levels are reduced due to inhibited synthesis or accelerated degradation, thereby directly affecting plant growth and yield30. DS significantly reduced SPAD values of the nonprimed seedlings. According31, It was found that under drought conditions, leaf chlorophyll content (SPAD) decreased due to the chloroplast damage caused by ROS. The different priming treatments enhanced the SPAD Value of NIL-SUB1DRO1 seedlings (Fig. 4e). Interestingly, osmo-priming significantly enhanced SPAD value in NIL-SUB1DRO1compared to halo and hydro priming methods under DS. This result is in line with findings reported by32. Enhanced chlorophyll content can contribute to improved photosynthetic efficiency and overall plant vigor33. These findings agree with the results obtained by33 who found that the SPAD value of rice seedlings was enhanced by osmo priming. A strong positive correlation was observed among SPAD, photosynthetic rate, maximum quantum yield, and biomass-related traits (e.g., root and shoot DW). This suggests that plants with higher chlorophyll content and photosynthetic efficiency exhibit greater carbon fixation and biomass accumulation under drought (Fig. 3c and d).

Compared to absolute water content, leaf relative water content is a good measure of cell and tissue hydration, crucial for optimal metabolic activity and a key drought tolerance indicator4. In the present study, the decline in water relation parameters of nonprimed seedlings under DS conditions agrees with previous findings of Zhang et al., Bai et al.32,34 reported decreased in relative water content of corn Zea mays L. and Sorghum bicolor L. Moench under drought stress conditions. However, priming significantly enhanced the relative water content and leaf water potential of the NIL-SUB1DRO1 genotype under DS (Fig. 4a & b). This result aligned with results reported by32. It was found that Osmo priming markedly enhances both the relative water content and leaf water potential compared to non-primed under DS (Fig. 4a, b). This suggests that primed seedlings efficiently maintain their water status through reduced water loss during DS. Kumar et al.35 reported that greater levels of compatible osmolytes may have allowed seedlings to maintain appropriate water potential, thus offsetting the adverse effect of water stress. These findings also support earlier results by Afrin36 who reported that seedlings from seeds subjected to osmo-priming and hydropriming showed improved water use efficiency and sustained growth compared to non-priming treatment under stress conditions. The positive linear correlation observed between leaf water potential and photosynthetic rate suggests that the net photosynthetic rate increases with increasing relative water content. Photosynthetic activity is usually dependent on stomatal conductance of the leaves and water movement through the xylem37. Plants primarily enhance water economy by reducing transpiration through partial stomatal closure, which decreases the photosynthetic rate, as CO2 is restricted from entering the mesophyll38. Our data indicated that drought-induced increase in the transpiration rate led to a decline in photosynthetic rate in seedlings of non-primed seeds. This effect could be attributed to reduced leaf expansion, impaired photosynthetic machinery, premature leaf senescence, chloroplast lipid oxidation, and altered structural pigments and proteins39. However, osmo-primed, haloprimed, and hydro-primed treatments alleviated the adverse effects of drought on the transpiration and photosynthetic rates of the NIL-SUB1DRO1 strain compared to non-primed seeds under DS (Fig. 4c, d). These results agree with the findings reported by40.

It is evident from the results that all the primed treatments enhanced the transpiration rate of NIL-SUB1DRO1, which correlated with a larger leaf area, suggesting that primed treatments can boost the plant roots’ ability under DS. Previous studies have indicated that the diminished photosynthetic activity of non-primed under DS could be attributed to stomatal or nonstomatal mechanisms6. Photosynthesis is typically inhibited during mild stress due to reduced CO2 diffusion, with stomatal conductivity decreasing before photosynthesis under drought conditions9. Moreover, seed priming helps preserve the photosystem structure and function, thereby enhancing overall photosynthetic performance. A severe DS in rice was revealed by reduced Photosynthetic rate. At the same drought level, different priming treatments showed less reduction in Photosynthetic rate than unprimed seedlings under DS. High photosynthetic capacity could be attributed to an increase in leaf chlorophyll content of NILSUB1DRO1 seedlings derived from primed treatments. A lower decrease in photosynthetic rate in primed seedlings suggested that these seedlings were more adaptive to DS and had the potential to maintain the plant functions. This result is supported by40 who also reported similar results. This aligns with the fact that photosynthetic activity and leaf chlorophyll content (SPAD) have a positive correlation41. Stomatal conductance reflects the extent of stomatal opening in a plant leaf, and greater conductance allows more water vapor and CO₂ to diffuse through it. High vapor pressure deficit (VPD), which characterizes dry air, promotes water evaporation from leaves into the atmosphere. Even if stomata remain partially closed, a high VPD may strengthen the diffusion gradient of water, resulting in an increased transpiration rate42. The dominance of PC1in (Fig. 5), suggests that the primary factor differentiating the samples is strongly related to the combined influence of root DW, photosynthetic rate, and Fv/Fm, along with the opposing influence of transpiration. This harmonious interplay highlights the fundamental significance of these parameters in promoting subsequent seedling development. The photosynthetic rate and SPAD values showed a positive correlation with maximum quantum yield, leaf area, relative water content, leaf water potential, and root and shoot DW (Fig. 6). The scatter plots in (Fig. 7), illustrated a positive linear relationship among SPAD, photosynthetic rate, and transpiration rate, indicating that the DS effect on these traits was diminished by the different priming treatments. However, a strong linear relationship in growth parameters under well-watered and DS highlights the importance of seed priming in enhancing these traits under DS. Thus, seed priming plays a fundamental role in the establishment of basal resistance to environmental stresses.

Conclusion

Drought stress (DS) reduced the growth performance of the non-primed seedlings, while the seed priming treatments enhanced the physiological performance of the NIL-SUB1DRO1 line with in the IR64 genetic background. Amongst these treatments, osmo priming with PEG was the most effective, in enhancing drought stress tolerance through positive effects on leaf area, SPAD value, (Fv/Fm), photosynthetic rate, shoot and root weight. These findings highlight osmo seed priming as a practical and promising seed priming technique in mitigating DS. Further studies focusing on biochemical mechanisms of seed priming on rice under drought stress is recommended.

Acknowledgements

The authors acknowledge the financial support from the Japan International Cooperation Agency (JICA) for the Agriculture Studies Networks for Food Security (Agri-Net) Scholarship that enabled the completion of this study. We also thank ENAGO (http://www.enago.jp) for the English language review.

Author contributions

A.T: Conceptualization, methodology, project administration, investigation, formal analysis, writing original draft, validation, visualization. J.I.S, and T.O: Conceptualization, methodology, resources, supervision, validation, writing, review & editing. A.L.L.W, I.S, E.O, A.M.I : Conceptualization data curation, data analysis, data validation review & editing. All authors read and approved of the final manuscript.

Funding

The Japan International Cooperation Agency (JICA) provided funding for this research.

Data availability

Data will be made available upon request from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Chun, Y. et al. MINI SEED 2 (MIS2) encodes a receptor-like kinase that controls grain size and shape in rice. Rice13, 1–17. 10.1186/s12284-020-0368-9 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Muthayya, S., Sugimoto, J. D., Montgomery, S. & Maberly, G. F. An overview of global rice production, supply, trade, and consumption. Ann. N Y Acad. Sci.1324, 7–14. 10.1111/nyas.12540 (2014). [DOI] [PubMed] [Google Scholar]
  • 3.Zheng, M. et al. Seed priming in dry direct-seeded rice: consequences for emergence, seedling growth and associated metabolic events under drought stress. Plant. Growth Regul.78, 167–178 (2016). [Google Scholar]
  • 4.Alam, A. U. et al. Seed priming enhances germination and morphological, physio-biochemical, and yield traits of cucumber under water-deficit stress. J. Soil. Sci. Plant. Nutr.23, 3961–3978. 10.1007/s42729-023-01314-3 (2023). [Google Scholar]
  • 5.Shao, H., Chu, L., Abdul, C. & Zhao, C. Water-deficit stress-induced anatomical changes in higher plants. 331, 215–225. 10.1016/j.crvi.2008.01.002 (2008). [DOI] [PubMed]
  • 6.Anjum, S. A. et al. Morphological, physiological and biochemical responses of plants to drought stress. 10, 1–7 (2020).
  • 7.Golzardi, F., Vazan, S., Moosavinia, H. & Tohidloo, G. Effects of salt and drought stresses on germination and seedling growth of swallow wort (Cynanchum acutum L). Res. J. Appl. Sci. Eng. Technol.4, 4524–4529 (2012). [Google Scholar]
  • 8.Apel, K. & Hirt, H. Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant. Biol.55, 373–399. 10.1146/annurev.arplant.55.031903.141701 (2004). [DOI] [PubMed] [Google Scholar]
  • 9.Farooq, M., Kobayashi, A. W., Fujita, N. & Basra, S. M. A. Review article plant drought stress: effects, mechanisms and management. Agron. Sustain. Dev.29, 185–212. 10.1051/agro:2008021 (2009). [Google Scholar]
  • 10.Venkateswarlu, B. & Shanker, A. K. Climate change and agriculture: adaptation and mitigation stategies. Ind. J. Agron.54, 226–230 (2009). [Google Scholar]
  • 11.Khan, S. et al. Development of drought-tolerant transgenic wheat: achievements and limitations. Int. J. Mol. Sci.20, 1–18. 10.3390/ijms20133350 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Pandey, V. & Shukla, A. Acclimation and tolerance strategies of rice (Oryza sativa L.) under drought acclimation and tolerance strategies of rice under drought stress. 630810.1016/j.rsci.2015.04.001 (2015). [Google Scholar]
  • 13.Kijoji, A. A. et al. Water extraction and root traits in Oryza sativa × Oryza glaberrima introgression lines under different soil moisture regimes water extraction and root traits in Oryza sativa ¾ Oryza glaberrima introgression lines under different soil moisture regimes. 10.1071/FP12163 (2012). [DOI] [PubMed]
  • 14.Uga, Y., Okuno, K. & Yano, M. Dro1, a major QTL involved in deep rooting of rice under upland field conditions. 62, 2485–2494 (2011). [DOI] [PubMed]
  • 15.Soe, I. et al. Assessing stress tolerance of SUBI and DRO1 introgression lines under flooding and drought conditions at different growth stages. 10.1101/2024.09.23.614591 (2025).
  • 16.Salam, A. et al. Nano-priming against abiotic stress: a way forward towards sustainable agriculture. 1–24 10.3390/su142214880 (2022).
  • 17.Sivritepe, N., Sivritepe, H. O. & Eris, A. The effects of NaCl priming on salt tolerance in melon seedlings grown under saline conditions. 97, 229–237 10.1016/S0304-4238(02)00198-X (2003).
  • 18.Baghizadeh, A. & Yazdanpanah, A. The effects of salicylic acid on some of biochemical and morphological characteristic of Ocimum basilicucm under salinity stress the effects of salicylic acid on some of biochemical and morphological characteristic of Ocimum basilicucm under salinity Stre. (2014).
  • 19.Artika, K. K. et al. ScienceDirect morpho-physiological response of Oryza glaberrima to gradual soil drying. 2710.1016/j.rsci.2019.12.007 (2020).
  • 20.Rouhi, H. R. & Aboutalebian, M. A. & Sharif-zadeh, F. Effects of hydro and osmopriming on drought stress tolerance during germination in four grass species. (2011).
  • 21.Mouradi, M. et al. Osmopriming improves seeds germination, Growth, antioxidant responses and membrane stability during early stage of Moroccan alfalfa populations under water deficit to cite this version : HAL id : hal-01389061 osmopriming improves seeds germination, Gro. 10.4067/S0718-58392016000300002 (2020).
  • 22.Yadav, S. P. S. et al. Effect of different chemical priming agents on physiological and morphological characteristics of rice (Oryza sativa L). Heliyon910.1016/j.heliyon.2023.e22389 (2023). [DOI] [PMC free article] [PubMed]
  • 23.Lee, S. S. & Kim, J. H. Rice Activity2.Pdf. Korean J. Crop Sci.45, 108–111 (2000). [Google Scholar]
  • 24.Hussain, M., Farooq, M. & Lee, D. J. Evaluating the role of seed priming in improving drought tolerance of pigmented and non-pigmented rice. J. Agron. Crop Sci.203, 269–276. 10.1111/jac.12195 (2017). [Google Scholar]
  • 25.Khalil, S. K. et al. Seed priming and phosphorus application enhance phenology and dry matter production of wheat. Pak J. Bot.42, 1849–1856 (2010). [Google Scholar]
  • 26.Sone, C., Ito, O. & Sakagami, J. Characterizing submergence survival strategy rice via chlorophyll fluorescence. 198, 152–160 (2012).
  • 27.Alharbi, B. M., Abdulmajeed, A. M. & Hassan, H. Biochemical and molecular effects induced by Triacontanol in acquired tolerance of rice to drought stress. Genes (Basel). 1210.3390/genes12081119 (2021). [DOI] [PMC free article] [PubMed]
  • 28.Saddiq, M. S. et al. Mitigation of salinity stress in wheat (Triticum aestivum L.) seedlings through physiological seed enhancements. J. Plant. Nutr.42, 1192–1204. 10.1080/01904167.2019.1609509 (2019). [Google Scholar]
  • 29.Jisha, K. C. & Puthur, J. T. Seed priming with beta-amino butyric acid improves abiotic stress tolerance in rice seedlings. Rice Sci.23, 242–254. 10.1016/j.rsci.2016.08.002 (2016). [Google Scholar]
  • 30.Muller, O. et al. Association between photosynthesis and contrasting features of minor veins in leaves of summer annuals loading phloem via symplastic versus apoplastic routes. Physiol. Plant. 152, (2014). [DOI] [PubMed]
  • 31.Yiu, J. C., Liu, C. W., Yi-Tan Fang, D. & Lai, Y. S. Waterlogging tolerance of Welsh onion (Allium fistulosum L.) enhanced by exogenous spermidine and spermine. Plant Physiol. Biochem.47, 710–716 (2009). [DOI] [PubMed] [Google Scholar]
  • 32.Zhang, F. et al. Seed priming with polyethylene glycol induces physiological changes in sorghum (Sorghum bicolor L. moench) seedlings under suboptimal soil moisture environments. PLoS One. 10, 1–15 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Dhillon, B. S. et al. Seed priming with potassium nitrate and gibberellic acid enhances the performance of dry direct seeded rice. (2021).
  • 34.Bai, L. P. et al. Effect of soil drought stress on leaf water status, membrane permeability and enzymatic antioxidant system of maize. Pedosphere16, 326–332 (2006). [Google Scholar]
  • 35.Kumar, S. G., Reddy, A. M. & Sudhakar, C. NaCl effects on proline metabolism in two high yielding genotypes of mulberry (Morus alba L.) with contrasting salt tolerance. Plant Sci.165, 1245–1251. 10.3390/agronomy11050849 (2003). [Google Scholar]
  • 36.Afrin, M. S. Osmo and hydro priming under drought stress condition stress condition by in agronomy. (2021).
  • 37.Liang, D. et al. Scientia horticulturae exogenous melatonin promotes biomass accumulation and photosynthesis of Kiwifruit seedlings under drought stress. Sci. Hortic.246, 34–43. 10.1016/j.scienta.2018.10.058 (2019). [Google Scholar]
  • 38.Bosabalidis, A. & Kofidis, G. Comparative effects of drought stress on leaf anatomy of two olive cultivars. Plant Sci.163, 375–379. 10.1016/S0168-9452(02)00135-8 (2002). [Google Scholar]
  • 39.Sgherri, C. Activated oxygen production and detoxification in wheat plants subjected to a water deficit programme. 10.1093/jxb/46.9.1123 (2018).
  • 40.Abid, M. et al. Seed osmopriming invokes stress memory against post-germinative drought stress in wheat (Triticum aestivum L). Environ. Exp. Bot.145, 12–20. 10.1016/j.envexpbot.2017.10.002 (2018). [Google Scholar]
  • 41.Sim, C. C., Zaharah, A. R., Tan, M. S. & Goh, K. J. Rapid determination of leaf chlorophyll concentration, photosynthetic activity and NK concentration of Elaies guineensis via correlated SPAD-502 Chlorophyll Index. 10.3923/ajar.2015.132.138.
  • 42.Serraj, R. et al. Drought-resistant rice: physiological framework for an integrated research strategy. In Drought Frontiers in Rice. 139–170 10.1142/9789814280013_0009 (World Scientific, 2009).

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data will be made available upon request from the corresponding author.

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

RESOURCES