Chloroplast-Derived Reactive Oxygen Species Dynamics Mediate Jasmonic Acid-Induced Drought Tolerance in Rice

doi:10.1016/j.rsci.2026.03.005

Abstract

Abstract:

Beyond their central role in photosynthesis and plant productivity, chloroplasts are emerging as key regulators of plant responses and tolerance to abiotic stress. However, the molecular mechanisms linking chloroplastic reactive oxygen species (ROS) metabolism to stress adaptation remain poorly understood. Here, we show that knockout of OsJAZ9, a repressor of jasmonic acid (JA) signaling, enhances JA response and drought tolerance in rice by suppressing cellular ROS accumulation. Time-lapse visualization of subcellular ROS dynamics revealed that stress-induced ROS accumulation was initiated in chloroplasts and subsequently propagated to the cytoplasm. Notably, loss of OsJAZ9 markedly suppressed chloroplastic ROS accumulation, resulting in reduced cellular ROS levels under stress conditions. Consistently, either JA treatment or overexpression of the JA-responsive chloroplastic ROS scavenger OsFeSOD3 was sufficient to attenuate chloroplastic ROS accumulation and enhance drought tolerance in rice. Furthermore, two-year agronomic field analyses showed that osjaz9 knockout rice exhibited improved grain yield, particularly under drought stress conditions. Together, our findings identify chloroplast-derived ROS dynamics as a pivotal molecular link between JA signaling and rice stress tolerance and highlight OsJAZ9 as a promising molecular target for developing high-yielding, stress-tolerant rice cultivars.

Key words: jasmonic acid, OsJAZ9, abiotic stress, reactive oxygen species, chloroplast, rice

Jiwoong Jung, Deok Hyun Seo, Youngdae Yoon, Geupil Jang. Chloroplast-Derived Reactive Oxygen Species Dynamics Mediate Jasmonic Acid-Induced Drought Tolerance in Rice[J]. Rice Science, 2026, 33(4): 531-544.

Figures/Tables 8

Fig. 1. osjaz9 knockout mutants exhibit improved grain yield under field conditions. A, Morphology of field-grown wild-type (WT) and osjaz9 knockout rice at the ripening stage in 2024 and 2025. Scale bars, 20 cm. B‒I, Quantification of growth and yield traits in WT and osjaz9 knockout plants in 2024 and 2025: plant height (B), panicle length (C), number of tillers per plant (D), number of spikelets per tiller (E), grain-filling rate (F), total grain number per plant (G), grain yield per plant (H), and 1000-grain weight (I). Data are mean ± SD (n > 30). Asterisks indicate statistically significant differences between WT and osjaz9 plants (*, P < 0.01, two-tailed t-test).

Fig. 2. Knockout mutation of OsJAZ9 enhances grain yield under drought stress conditions. A, Whole-plant images of wild-type (WT) and osjaz9 knockout rice at the harvest stage under drought stress. Scale bar, 20 cm. B, Grain images showing clear differences in grain-filling rates between WT and osjaz9 rice. Scale bar, 1 cm. C‒J, Analysis of agronomic traits in WT and osjaz9 knockout plants: plant height (C), panicle length (D), number of tillers per plant (E), number of spikelets per tiller (F), grain-filling rate (G), total grain number per plant (H), grain yield per plant (I), and 1000-grain weight (J) in 2024 and 2025. Data are mean ± SD (n > 30). Asterisks indicate statistically significant differences between WT and osjaz9 plants (*, P < 0.01, two-tailed t-test).

Fig. 3. Yield advantage of osjaz9 rice under drought conditions is largely due to improved grain-filling rate. Spider plots summarizing differences in agronomic traits between wild-type (WT) and osjaz9 knockout rice plants grown under normal and drought stress conditions over two consecutive years (2024 and 2025; n > 30 for each year). Values are expressed as percentages relative to the WT mean (set to 100%).

Fig. 4. Suppression of photosynthetic performance under drought stress is attenuated in OsJAZ9 knockout mutants. A and B, Drought tolerance assay of wild-type (WT) and osjaz9 knockout plants at the early vegetative stage (A) and the booting stage (B). Three-week-old (A) and 10-week-old (B) WT and osjaz9 plants grown under normal conditions (control) were subjected to drought stress (drought) for 3 d. Following drought treatment, the plants were re-watered and allowed to recover (re-watering) under normal growth conditions for 10 d. Scale bars, 5 cm in A and 20 cm in B. C‒E, Quantification of photosynthetic parameters in drought-tested WT and osjaz9 plants at the booting stage: total chlorophyll content (C), transcript levels of OsRbcL (D) in leaves, and maximum quantum yield of PSII (Fv/Fm, n > 60) (E). Chlorophyll content and Fv/Fm data represent means of three biological replicates, and qRT-PCR data represent means of nine replicates (three biological × three technical). OsTUB2 was used as an internal control to normalize qRT-PCR results. Error bars indicate SD. Asterisks indicate statistically significant differences between WT and osjaz9 plants (*, P < 0.01, two-tailed t-test).

Fig. 5. Cellular reactive oxygen species (ROS) accumulation is reduced by OsJAZ9 knockout mutation. A and B, 3,3ʹ-Diaminobenzidine (DAB, A) and nitroblue tetrazolium (NBT, B) staining of wild-type (WT) and osjaz9 plants under normal and drought conditions. Three-week-old plants were subjected to drought stress for 3 d. Scale bars, 0.5 cm. C, Quantification of NBT staining in WT and osjaz9 plants by measuring formazan content. Formazan levels were determined at A630 and normalized to fresh weight. Data are mean ± SD (n = 3). Asterisks indicate statistically significant differences between WT and osjaz9 plants under drought conditions (*, P < 0.01, two-tailed t-test). D, 2ʹ,7ʹ-Dichlorodihydrofluorescein diacetate (H2DCFDA) staining and confocal microscopic observation in WT and osjaz9 plants. Blue and red fluorescence correspond to H2DCFDA and chlorophyll autofluorescence signals, respectively. Scale bars, 10 µm.

Fig. 6. Knockout mutation of OsJAZ9 suppresses chloroplastic reactive oxygen species accumulation under stress conditions. A and B, Time-lapse monitoring of 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence in protoplasts isolated from 1-week-old wild-type (WT) and osjaz9 plants. Protoplasts were treated with 1.5 mmol/L H2O2 (A) or co-treated with 1.5 mmol/L H2O2 and 1.0 mmol/L N-acetyl-L-cysteine (NAC) (B) and incubated for the indicated times (0, 1, 2, 4, and 6 h). Blue and red fluorescence indicate H2DCFDA and chlorophyll autofluorescence signals, respectively. Scale bars, 10 µm.

Fig. 7. Jasmonic acid (JA) enhances drought tolerance by reducing cellular reactive oxygen species (ROS) levels. A, Drought stress tolerance of 3-week-old rice plants treated or untreated with JA. Plants were treated with mock (MOCK) or 100 μmol/L JA for 12 h and subjected to drought stress for 3 d. Plants were then re-watered and grown under normal conditions for 10 d to assess recovery. Scale bars, 5 cm. B and C, Quantification of photosynthetic parameters in MOCK and JA rice plants under drought stress: total chlorophyll content (B) and transcript levels of OsRbcL (C). D, Survival rates of MOCK and JA rice plants after re-watering. Survival rate was calculated as the number of plants surviving after re-watering divided by the total number of plants tested (n > 128). E, Nitroblue tetrazolium (NBT) staining showing ROS accumulation in MOCK and JA rice plants under normal (control) and drought stress conditions. Scale bars, 0.5 cm. F, Expression patterns of chloroplastic ROS-scavenging genes (OsAPX7, OsAPX8, OsFeSOD2, and OsFeSOD3) in MOCK and JA rice leaves. Chlorophyll content data represent means of three biological replicates, and qRT-PCR data represent means of nine replicates (three biological × three technical). OsTUB2 was used as an internal control to normalize qRT-PCR results. Error bars indicate SD. Asterisks indicate statistically significant differences compared with the corresponding controls (*, P < 0.01, two-tailed t-test).

Fig. 8. Overexpression of jasmonic acid (JA)-responsive OsFeSOD3 enhances drought tolerance by attenuating chloroplastic reactive oxygen species (ROS) accumulation. A, Phenotypes of wild-type (WT) and two independent OsFeSOD3-overexpressing transgenic lines (Ox #1 and #2) under drought stress. Plants were grown under normal conditions for 3 weeks, and then exposed to drought stress for 3 d. Following drought treatment, these plants were re-watered and allowed to recover under normal growth conditions for 10 d. Scale bars, 5 cm. B, Chloroplastic ROS accumulation visualized by 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (H2DCFDA) staining in WT and OsFeSOD3-overexpressing plants under drought- untreated (control) and -treated (drought) conditions. Blue and red fluorescence indicate H2DCFDA and chlorophyll autofluorescence, respectively. Scale bars, 5 µm. C, Time-course H2DCFDA imaging of cellular and chloroplastic ROS dynamics in WT and OsFeSOD3-overexpressing rice plants under stress. Protoplasts isolated from 1-week-old WT and OsFeSOD3-overexpressing plants were treated with 1.5 mmol/L H2O2 and incubated for indicated times (0, 1, 2, 4, and 6 h). Chloroplastic and cellular ROS were visualized by H2DCFDA fluorescence (blue), and chlorophyll autofluorescence is shown in red. Scale bars, 10 µm.

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