Effects of Drought Stress and Rewatering on the Photosynthetic Physiology of Rice Seedlings ()
1. Introduction
Global climate change is increasing both the frequency and intensity of extreme drought events, thereby exacerbating abiotic constraints on crop production systems [1] [2]. Drought disrupts plant water balance, suppresses key metabolic processes and impedes growth and development. It has become one of the major factors limiting crop yield and quality, and a serious threat to global food security. Dissecting the physiological mechanisms by which crops respond to drought stress is therefore critical for designing climate resilient agricultural strategies and ensuring sustainable grain production [3] [4].
Rice is the staple food for more than half of the world’s population, and the drought resilience of rice production systems is of paramount importance. However, due to its physiological characteristics, rice is highly sensitive to water deficit [5] [6]. Drought frequently impairs its photosynthetic machinery, reducing assimilate accumulation and ultimately yield. In particular, understanding how the photosynthetic apparatus, especially photosystem II (PSII)—responds dynamically to drought and subsequent rewatering is essential for clarifying the physiological basis of drought tolerance and for guiding drought resilient breeding and cultivation practices.
Photosynthesis provides the material and energy foundation for crop yield formation. PSII is the primary site of photochemical charge separation and the initiation point of the photosynthetic electron transport chain, and is also one of the components most sensitive to environmental stress [7] [8]. Chlorophyll fluorescence techniques, especially rapid chlorophyll a fluorescence induction kinetics (OJIP transients) and the associated JIP‑test, allow noninvasive and real-time monitoring of the full sequence of events in PSII reaction centers, from light absorption to electron transport and energy conversion. By analyzing key fluorescence parameters such as the maximum quantum efficiency of PSII (Fv/Fm), the performance index (PIabs) and the energy fluxes per reaction center (e.g. ABS/RC, TRo/RC, ETo/RC), the functional status of the donor side, reaction center and acceptor side of PSII can be precisely diagnosed. These tools have become powerful methods for evaluating plant stress physiology and for early detection of environmental constraints [9] [10].
Previous studies have shown that drought stress affects PSII function in rice through multiple mechanisms. It can initially impair the activity of the oxygen evolving complex (OEC), thereby hampering water splitting and electron donation [11] [12]. Drought may also cause reversible inactivation or irreversible damage of PSII reaction centers, leading to blockage of electron transport. In addition, drought disturbs excitation energy allocation, enhances non-photochemical quenching and induces photoinhibition [13] [14]. At the fluorescence level, these lesions are typically manifested as decreases in Fv/Fm and PIabs, accompanied by increases in ABS/RC and TRo/RC, which often reflect a reduction in the number of active reaction centers and a higher excitation burden on those remaining [15]. Rewatering offers an opportunity to assess the repair capacity of the photosynthetic apparatus; however, part of the drought‑induced damage may be irreversible, and the rate and extent of recovery are key indicators of varietal drought tolerance. Although OJIP‑based approaches have been applied to evaluate drought tolerance in rice, most studies have focused on single stress levels or specific growth stages and have often been limited to static observations. Systematic investigations of the dynamic responses and recovery patterns of PSII function in rice seedlings under progressive soil drying and subsequent rewatering remain scarce. In particular, a quantitative dissection of the energy fluxes through discrete PSII modules using the JIP‑test, integrated with morphological and physiological traits to explain varietal differences in drought tolerance, is still lacking [16]-[19].
In this study, rice cultivars with contrasting drought tolerance were grown in pots under controlled water regimes to simulate progressive soil drought followed by rewatering. By dynamically recording OJIP transients and conducting JIP-test analysis, we aimed to: 1) characterize the temporal dynamics and damage sites of PSII light harvesting, electron transport, and energy conversion under drought stress; 2) evaluate the recovery capacity and limiting factors of PSII function after rewatering; and 3) elucidate the photosynthetic physiological mechanisms underlying cultivar-specific differences in drought responses. This work seeks to deepen our understanding of drought stress and subsequent recovery in rice seedlings from the perspective of photosynthetic energy fluxes, while providing sensitive physiological indicators and theoretical support for drought tolerance assessment and genetic improvement of rice. To further clarify the photosynthetic and physiological responses of rice seedlings to drought stress and rewatering, four rice cultivars (HHZ, PH34, WGZ, and LD24) were employed as experimental materials. Three water treatments were applied, including a well-watered control (CK), drought stress (DS), and rewatering (RW) following drought exposure. This study focused on leaf chlorophyll content and quantified variations in photosystem II (PSII) functionality using the JIP-test to analyze rapid chlorophyll a fluorescence induction kinetics (OJIP transients).
2. Materials and Methods
2.1. Experimental Materials and Design
The experiment was carried out in the Smart Crop Factory of the College of Agriculture, Fujian Agriculture and Forestry University, under controlled environmental conditions with an average temperature of 28˚C and relative humidity of 70%. Four rice cultivars, namely HHZ, PH34, WGZ, and LD24, were provided by the Fujian Provincial Engineering Technology Research Center for Special Crop Breeding and Utilization. Paddy soil (moisture content: 31.00%, pH ≈ 5.6) was used as the base substrate, and a mixture of paddy soil and commercial nutrient soil at a ratio of 3:1 was prepared for seedling transplantation. To clarify the photosynthetic and physiological responses of rice seedlings to drought stress and subsequent rewatering, these four cultivars were employed as experimental materials. Three water treatments were applied, including a well-watered control (CK), drought stress (DS), and rewatering (RW) following drought exposure. This study focused on leaf chlorophyll content and quantified variations in photosystem II (PSII) functionality using the JIP-test to analyze rapid chlorophyll a fluorescence induction kinetics (OJIP transients).
Three water treatments were imposed: well watered control (normal, moisture content is maintained at 80% ± 5% of the field water-holding capacity), drought stress (DS, moisture content is maintained at 40% ± 5% of the field water-holding capacity) and rewatering. For the drought treatment, irrigation was stopped when the seedlings had recovered from transplanting and reached the four leaf one heart stage. Plants were monitored daily between 11:00 and 12:00. When a portion of leaves exhibited visible drought symptoms yellowing, wilting and curling the plants were considered to have reached the drought stress point. At this time, measurements were taken, after which the pots were thoroughly irrigated to initiate the rewatering treatment, and no further water was supplied thereafter. Morphological indices were measured again 5 days after rewatering.
2.2. Measurements and Methods
2.2.1. Determination of Morphological Parameters
From each treatment, three pots were randomly sampled, and three uniformly growing seedlings with similar vitality were measured per pot. Plant height, leaf length and leaf width were recorded, and leaf area was estimated by applying the leaf area coefficient.
2.2.2. SPAD Readings and Chlorophyll Content
Relative chlorophyll content was assessed using a SPAD 502 chlorophyll meter (Konica Minolta, Japan). For each treatment, three plants with uniform growth were selected per pot. On each plant, four leaves were chosen, and three positions per leaf were measured three times; the mean SPAD value was recorded.
For total chlorophyll, 0.2 g of functional leaf tissue was rapidly sampled and placed into a 10 mL centrifuge tube. An appropriate volume of 95% ethanol was added to fully immerse the leaves. Samples were extracted in darkness at 4˚C for 48 h, with intermittent shaking to ensure complete pigment extraction. Absorbance of the extracts was determined at 665 nm and 649 nm using a spectrophotometer. Each sample was measured in triplicate, and mean values were used to calculate total chlorophyll content according to standard equations.
2.2.3. OJIP Transients and Chlorophyll Fluorescence Parameters
Chlorophyll fluorescence was measured using a Handy PEA portable plant efficiency analyzer (Hansatech Instruments, UK). Prior to measurement, leaves were dark‑adapted for 30 min. For each treatment, three uniformly growing plants were selected per pot, and four leaves per plant were measured. On each leaf, three positions were selected and measured three times.
OJIP transients were recorded, and the following fluorescence parameters were obtained via JIP‑test: initial fluorescence (Fo), maximum fluorescence (Fm), variable fluorescence (Fv) and derived ratios Fo/Fm, Fv/Fm and Fv/Fo; Vj and Sm, which reflect electron transport characteristics; energy fluxes per reaction center (ABS/RC, TRo/RC, ETo/RC, DIo/RC); energy fluxes per cross‑section (TRo/CSo, ETo/CSo, DIo/CSo, REo/CSo); and comprehensive performance indices (PIabs, PItotal). These parameters include both directly measured raw fluorescence values and physiological parameters calculated from them.
To further dissect PSII behavior at different phases of the fluorescence induction, the OJIP curves were normalized at multiple time points: 50 μs (O step), 300 μs (K step), 2 ms (J step), 30 ms (I step) and the time of maximum fluorescence (P step). This multi‑time‑point normalization was used to analyze PSII light energy utilization and electron transport characteristics at distinct stages.
2.3. Data Processing and Statistical Analysis
Data were analyzed using IBM SPSS Statistics 27. One way ANOVA was performed followed by LSD tests to assess significant differences among treatments. Figures were generated using Microsoft Excel 2016 and Origin 2025.
3. Results and Analysis
3.1. Effects of Drought Stress and Rewatering on Morphological
Parameters of Rice Seedlings
The dynamic changes of morphological indexes of rice after CK (normal), drought and rehydration treatment are shown in Table 1.
Drought treatment caused declines in leaf length, leaf width, leaf area, and plant height across all rice materials, which recovered after rehydration. As shown in Table 1, the drought tolerance varied significantly among materials. Compared to the control group (CK), all four rice materials exhibited reduced leaf length, width, and plant height post-drought. Following rehydration, all four materials showed rebounding increases in leaf length, width, plant height, and leaf area, with the experimental material WGZ demonstrating the most pronounced recovery in leaf length and width. Overall, drought treatment significantly affected leaf dimensions and plant height, but rehydration restored all morphological traits, though the recovery varied among drought-tolerant rice types.
Table 1. Effects of drought stress and rewatering on morphological parameters of rice seedlings.
Material |
Treatment |
Morphological indices (
) |
Leaf length |
Leaf width |
Leaf area |
Plant height |
HHZ |
CK |
25.68 ± 3.63a |
0.64 ± 0.02a |
13.70 ± 2.82a |
35.47 ± 4.39a |
|
Drought stress |
13.23 ± 1.58c |
0.36 ± 0.04c |
4.36 ± 1.22c |
27.63 ± 3.11b |
Rewatering |
19.34 ± 5.67b |
0.38 ± 0.05b |
7.82 ± 2.11b |
30.05 ± 3.15b |
PH34 |
CK |
25.86 ± 1.97a |
0.61 ± 0.02a |
13.25 ± 1.75a |
35.59 ± 2.12a |
Drought stress |
14.34 ± 2.08b |
0.36 ± 0.01c |
4.63 ± 0.87c |
27.78 ± 3.00ab |
Rewatering |
20.49 ± 5.13c |
0.46 ± 0.04b |
8.05 ± 1.41b |
30.34 ± 2.13b |
WGZ |
CK |
16.51 ± 2.17b |
0.52 ± 0.02ab |
7.49 ± 0.92b |
28.79 ± 1.33a |
Drought stress |
14.28 ± 2.11b |
0.49 ± 0.03b |
6.36 ± 1.11b |
25.96 ± 2.03a |
Rewatering |
21.60 ± 2.97a |
0.56 ± 0.03a |
10.32 ± 1.35a |
27.30 ± 2.36a |
LD24 |
CK |
22.99 ± 1.45a |
0.55 ± 0.08a |
10.89 ± 2.66a |
27.96 ± 3.55a |
Drought stress |
17.54 ± 2.43b |
0.28 ± 0.09c |
4.53 ± 2.16b |
25.55 ± 1.58b |
Rewatering |
17.58 ± 1.45b |
0.42 ± 0.02b |
6.52 ± 1.01b |
26.70 ± 1.44b |
Note: In the same column, different lowercase letters denote statistically significant disparities among various treatment groups at the identical time point (P < 0.05). All data are expressed as mean ± standard error with three replicates (n = 3); The letters used for comparison are not applicable across distinct test materials. Abbreviations: CK: control group, DS: drought stress treatment, RW: rewatering treatment.
3.2. Effects of Drought and Rewatering on Relative and Absolute
Chlorophyll Content in Rice Leaves
As shown in Figure 1, chlorophyll content declined after drought treatment in all cultivars, with WGZ exhibiting the smallest reduction. Rewatering led to partial recovery of chlorophyll content, but in none of the cultivars did levels return to those of the well watered control. Among the cultivars, HHZ showed the greatest recovery after rewatering, with chlorophyll content increasing by 28.75% relative to the drought‑stressed level. LD24 was more strongly affected by drought, and its chlorophyll content after rewatering remained lower than under drought alone. Compared with CK, SPAD values in the four cultivars decreased by 17.09%, 12.52%, 11.61% and 15.85%, respectively, following drought stress. After rewatering, SPAD values partially recovered but remained below the control in all cases. These results indicate that drought stress causes substantial damage to leaf chlorophyll apparatus in rice, as reflected by reduced SPAD readings.
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Note: Different lowercase letters above the bars in the bar charts indicate significant differences (P < 0.05) among treatment combinations based on Duncan’s test. Values are presented as mean ± SE (n = 3). The error bars denote standard deviation. CK: Control. DS: Drought stress. RW: Rewatering.
Figure 1. Effects of drought stress and rewatering on relative and absolute chlorophyll content in rice leaves.
3.3. Effects of Drought Stress and Rewatering on OJIP Transients
in Rice
The dynamic changes in OJIP transients of rice leaves under CK, drought and rewatering treatments are shown in Figure 2. Rapid chlorophyll fluorescence represents the time‑dependent changes in fluorescence intensity emitted by chlorophyll a molecules after dark adapted leaves are exposed to strong actinic light, and serves as an effective probe of PSII reaction center activity and electron transport efficiency.
In this study, OJIP transients were recorded from fully dark‑adapted leaves and the corresponding parameters were analyzed. All treatments displayed typical OJIP induction curves. Fluorescence intensity at the O step (50 μs) was similar among treatments, with negligible differences. As induction progressed to the J step (2 ms), fluorescence in CK, drought and rewatering groups increased, and intensities at the I and P steps were substantially higher than at the O and J steps. Overall, the rise in the O-J segment was more gradual than in the I-P segment. Relative to CK, however, the J-I and I-P phases in the drought and rewatering groups exhibited a markedly slower increase and a reduced amplitude. For all four cultivars, fluorescence intensity from the I to P steps was significantly lower under drought and rewatering than in CK, with the overall decline in the OJIP curves being most pronounced under drought stress. These observations indicate that drought stress causes pronounced damage to the photosynthetic system of rice, reducing light energy conversion efficiency and suppressing photosynthesis. Although rewatering partially alleviated the effects of drought, photosynthetic function did not fully recover to control levels. Notably, in PH34 and WGZ, the rise from O to J was higher under rewatering than in CK, likely reflecting a transient photosynthetic compensation effect once favorable water conditions were restored.
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Figure 2. Changes in rapid chlorophyll fluorescence induction kinetics (OJIP transients) in rice under drought stress and rewatering.
To further elucidate the mechanisms underlying treatment‑dependent differences in fluorescence dynamics and to compare responses among phases of the OJIP transient, we performed differential analyses of the OJIP curves for two contrasting cultivars, HHZ and LD24. This approach facilitates detection of subtle changes in transition regions of the curve and provides a basis for evaluating the effects of drought stress and rewatering on photosynthetic function.
In OJIP based studies, the L‑, K‑ and J‑bands in differential curves are widely used to infer structural and functional changes in PSII. The L‑band primarily reflects the structural integrity of thylakoid membranes and the degree of energetic connectivity among PSII units. The K‑band is closely associated with the functional status of the OEC, while the J‑band indicates the efficiency of electron transport from the primary quinone acceptor QA to the secondary acceptor QB on the PSII acceptor side. Together, these indices provide important tools for assessing drought tolerance and recovery potential in rice.
As shown in Figure 3, ΔWL (L‑band), ΔWK (K‑band) and ΔWJ (J‑band) all increased to varying degrees in HHZ and LD24 following drought and rewatering, indicating disrupted energy transfer across thylakoid membranes and impaired connectivity among PSII reaction centers. The K‑band increased more strongly in HHZ than in LD24, suggesting more severe OEC damage and more pronounced restriction of electron donation from water. Both cultivars exhibited a pronounced J‑band, implying that under drought and rewatering, reduced QA (QA−) was not efficiently re‑oxidized by QB, leading to accumulation of QA− and decreased electron transport efficiency on the PSII acceptor side.
Figure 3. Differential changes in ΔWL (L‑band), ΔWK (K‑band) and ΔWJ (J‑band) in rice leaves under drought stress and rewatering.
3.4. Effects of Drought Stress and Rewatering on Chlorophyll
Fluorescence Kinetic Parameters
To gain deeper insight into how drought and rewatering affect the photosynthetic electron transport chain, we applied JIP‑test analysis to derive fluorescence kinetic parameters, including Fv/Fm, Vj, Sm, PIabs, PItotal, TRo/CSo, ETo/CSo, DIo/CSo and REo/CSo. These parameters provide a comprehensive view of PSII electron transport under different treatments. As shown in Figure 4, the relative variable fluorescence at the J step (Vj) tended to decrease under drought and rewatering. This pattern reflects perturbations in PSII reaction center function, likely involving impaired electron transfer from QA to QB and consequent closure of reaction centers. Drought stress may also compromise OEC activity, suppressing water photolysis and limiting electron supply, so that QA− cannot be re‑oxidized efficiently.
Across drought‑related treatments, the four cultivars exhibited significantly lower Fv/Fm, PIabs and PItotal compared with the control. Fv/Fm is a widely used indicator of stress severity; its decline indicates that drought strongly inhibits the normal operation of the photosynthetic apparatus, reducing the overall accumulation of photosynthates. Analysis of the energy flux parameters reflecting PSII reaction center activity (TRo/CSo, ETo/CSo, DIo/CSo, REo/CSo) showed that TRo/CSo and ETo/CSo decreased in all four cultivars under drought, demonstrating reduced efficiency of light energy capture and conversion into electron transport, as well as impaired electron flow from PSII to PSI. These changes likely disrupt ATP and NADPH production and lead to structural damage in the photosynthetic reaction centers. The decline in the comprehensive performance index PItotal further supports this conclusion. Overall, drought stress exerts multi‑faceted damage on PSII structure and function, substrate supply and electron transport. Although rewatering partially restored photosynthetic activity, PSII function did not fully return to the control state, indicating that some drought‑induced lesions are persistent.
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Note: (a) represent rice materials HHZ; (b) represent rice materials PH34; (c) represent rice materials WGZ; (d) represent rice materials LD24.
Figure 4. Dynamic changes in chlorophyll fluorescence kinetic parameters in rice leaves under drought stress and rewatering.
3.5. Effects of Drought Stress and Rewatering on the Status of PSII
Reaction Centers
As shown in Figure 5, the parameters the energy absorbed per reaction center (ABS/RC), the energy dissipated per reaction center (DIo/RC), the energy used for electron transport (ETo/RC), and the energy trapped per reaction center (TRo/RC) describe the energy partitioning per PSII reaction center. With increasing drought severity, all four cultivars showed rising trends in ABS/RC, TRo/RC, ETo/RC and especially DIo/RC. This indicates that the absorbed light energy, trapped energy, electron transport and dissipated energy per active reaction center increased under drought and rewatering.
(a) (b)
(c) (d)
Note: (a) Representing rice materials HHZ; (b) Representing rice materials PH34; (c) Representing rice materials WGZ; (d) Representing rice materials LD24.
Figure 5. Changes in PSII reaction center energy fluxes in rice leaves under drought stress and rewatering.
In combination with Figure 6, which shows specific activity parameters and RC/CSm (the density of active PSII reaction centers per excited thylakoid cross‑ section), it is evident that RC/CSm decreased under drought. Concomitant increases in ABS/RC, TRo/RC, ETo/RC and DIo/RC, together with decreases in RC/CSm and PIabs, indicate an increase in the proportion of inactivated reaction centers and a higher excitation load on the remaining active centers on a thylakoid cross‑section basis. In other words, drought stress reduces the number of functional PSII units, forcing the surviving centers to process more energy, which enhances the risk of photodamage. Rewatering partially alleviated these effects but did not fully restore PSII reaction center function.
Note: (a) Representing rice materials HHZ; (b) Representing rice materials PH34; (c) Representing rice materials WGZ; (d) Representing rice materials LD24.
Figure 6. Specific activity parameters, reaction center density RC/CSm and performance index based on absorbed energy in rice leaves under different drought treatments.
4. Discussion
Current rice cultivation practices predominantly rely on continuous flooding, which confers relatively poor drought tolerance, high water consumption and high vulnerability to drought and heat extremes [20] [21]. Numerous studies have demonstrated that drought stress causes pronounced physiological damage in rice, affecting morphological development, chlorophyll biosynthesis and reaction center activity, thereby markedly suppressing photosynthesis [22]. These physiological perturbations are ultimately reflected in plant morphology. Under drought, rice leaves typically curl and yellow. Moderate leaf rolling can help reduce transpiration and excessive light interception, representing an adaptive self‑protection strategy. Traits such as leaf size, trichome density, stomatal density and stomatal aperture are also important indicators of drought tolerance. In this study, drought stress significantly inhibited increases in plant height, leaf length, leaf width and leaf area. After rewatering, these morphological traits partially recovered, but the extent of recovery differed among cultivars.
Photosynthesis is a central metabolic process underpinning plant survival, growth, reproduction and ecosystem functioning. Under drought stress, photosynthesis is often the earliest and most strongly suppressed physiological process [23]. Chlorophyll, the key pigment for light harvesting and energy conversion, directly drives the light reactions that generate ATP and NADPH [24]. Under water deficit, chlorophyll content typically declines due to inhibited biosynthesis and accelerated degradation. Consistent with this general pattern, our results showed reduced chlorophyll content after drought, with only partial recovery after rewatering and chlorophyll levels remaining below those of well‑watered plants.
Photosynthesis comprises light reactions and carbon fixation reactions. In the light reactions, PSI and PSII cooperate to convert light energy into chemical energy and evolve oxygen [25]. OJIP transients sensitively reflect changes in the primary photochemical reactions of PSII reaction centers. The characteristic phases of the OJIP curve are closely related to the functional status of reaction centers, the activity of the electron transport chain and the integrity of the photosynthetic apparatus as a whole. Drought stress often first targets PSII, disrupting both the donor and acceptor sides and causing imbalances in electron transport. In this study, the amplitude of the OJIP rise under drought was lower than that of the control, indicating restricted electron transport. Under stress, characteristic L‑, K‑ and J‑band frequently appear, reflecting OEC damage, interruption of donor‑side electron flow, excess excitation energy and imbalances in the electron transport chain. These conditions enhance non‑photochemical energy dissipation, and PSI may also be secondarily impaired, leading to global inhibition of photosynthesis. The appearance of these characteristic bands after drought in our experiment indicates that drought disturbed electron flow on both sides of the PSII reaction center, causing structural damage.
Chlorophyll fluorescence parameters provide quantitative measures of energy partitioning and electron transport efficiency in the photosynthetic apparatus and are widely used to evaluate stress damage and tolerance mechanisms [26] [27]. Drought‑induced damage to the photosynthetic system is often concentrated on the PSII acceptor side, where electron transport is hindered, key components are inactivated and reactive oxygen species accumulate, causing oxidative damage and leading to declines in Fv/Fm, Fv/Fo and related parameters，among which HHZ showed the most significant decreasing trend. In our study, drought increased Vj and decreased Fv/Fm in rice, indicating elevated reduction pressure in the plastoquinone (PQ) pool on the PSII acceptor side and restricted electron transfer from QA to QB. The performance index based on absorption (PIabs) is a comprehensive indicator of the functional status of the photosynthetic apparatus; its reduction signifies structural or functional damage. Under drought stress, both PItotal and PIabs showed a decreasing trend across the four varieties, among which the decreasing trend of WGZ was the least significant. However, the extent of recovery is constrained by the severity and duration of prior stress. Here, severe drought markedly impaired the photosynthetic machinery and reaction centers of rice. Although leaf morphology partially recovered after rewatering, chlorophyll fluorescence traits indicated that the photosynthetic system retained persistent functional impairment.
5. Conclusion
In summary, drought stress suppresses photosynthesis in rice by reducing chlorophyll content, damaging PSII donor and acceptor sides, decreasing reaction center density and disturbing energy distribution, which together impair growth and development. Rewatering elicits partial compensatory responses but cannot fully reverse structural or severe functional damage to the photosynthetic apparatus. These findings provide a physiological basis for improving water use efficiency and drought resilience in rice under water‑limited conditions. Given the complexity of natural environments and the diversity of plant stress responses, further work is needed to refine our understanding of the underlying mechanisms and to translate these insights into robust agronomic and breeding strategies.