The OsAP4-OsCATA/OsCATC Regulatory Module Orchestrates Drought Stress Adaptation in Rice Seedlings Through ROS Scavenging

Abstract

Drought stress poses a major constraint on global crop productivity. Although aspartic proteases (APs) are primarily characterized in plant disease resistance, their roles in abiotic stress adaptation remain largely unexplored. Here, we demonstrate that rice (Oryza sativa) OsAP4 critically regulates drought stress tolerance at the seedling stage. Genetic manipulation through overexpression (OsAP4-OE) or CRISPR knockout (OsAP4-KO) resulted in significantly reduced or enhanced stress tolerance compared to wild-type plants, respectively. Through integrated approaches including yeast two-hybrid, bimolecular fluorescence complementation, pull-down, co-immunoprecipitation, and protein degradation assays, we established that OsAP4 physically interacts with and destabilizes OsCATA/OsCATC, two catalase enzymes responsible for reactive oxygen species (ROS) scavenging. Importantly, OsAP4 modulates ROS production under drought stress treatment conditions. Together, these findings reveal a novel OsAP4-OsCATA/OsCATC regulatory module governing rice drought stress responses.

Keywords: OsAP4; OsCATs; ROS scavenging; drought stress; rice.

Figure 1

Expression of OsAP4 under abiotic stress conditions. (A–D) OsAP4 expression under PEG, submergence, NaCl, and heat stress treatments from 0–24 h. Data are means ± SD (n = 3). (E) Evolutionary relationships of ASPG1 and five homologous aspartic proteinase family members of rice. (F) Expression of the five homologues under control and PEG stress treatment conditions. Data are means ± SD (n = 3); ns, not significant; ** p < 0.01 determined by t-test; the test was performed on samples between the control and PEG treatment.

Figure 2

Analysis of drought stress tolerance in OsAP4 transgenic plants. (A) Plant phenotypes, (B) relative seedling height, (C) relative biomass, and (D) survival rates of wild-type 9311 and OsAP4 overexpression lines under PEG-treated conditions. (E) Plant phenotypes, (F) relative seedling height, (G) relative biomass, and (H) survival rates of 9311 and OsAP4 knockout lines under PEG-treated conditions. In (B–D,F–H), data are means ± SD (n ≥ 5); ** p < 0.01 determined by t-test; the test was performed on samples between the 9311 and OE/KO lines. Bars = 2 cm in (A,E).

Figure 3

Analysis of candidate OsAP4-interacting proteins. (A) Expression of OsCATA/OsCATC in shoot and root of the plants under drought-mimicking type (DMT) from 0 to 12 h. (B,C) Expression of OsCATA/OsCATC in 9311 and OsAP4 transgenic plants under PEG treatment conditions. Data are means ± SD (n = 3); ** p < 0.01 determined by t-test; the test was performed on samples between the 9311 and OE/KO lines. (D) Subcellular localization of OsAP4 and OsCATA/OsCATC in N. benthamiana leaf cells. Bars = 20 μm.

Figure 4

OsAP4 interacts with OsCATA/OsCATC and reduces their stability. (A) Validation of OsAP4 interactions with OsCATA/OsCATC by Y2H assay. (B) Validation of OsAP4 interactions with OsCATA/OsCATC by BiFC assay. Bars = 20 μm. (C) Validation of OsAP4 interactions with OsCATA by pull-down assay. (D,E) Validation of OsAP4 interactions with OsCATA/OsCATC by Co-IP assay. (F,G) Validation of OsAP4 mediates OsCATA/OsCATC stability.

Figure 5

ROS content in OsAP4 and OsCATA/OsCATC transgenic plants. (A–D) DAB staining, FRAP, CAT activity, and MDA content in leaves of 9311 and OsAP4 overexpression plants under control and PEG stress conditions. (E–H) DAB staining, FRAP, CAT activity, and MDA content in leaves of 9311 and OsAP4 knockout plants under control and PEG stress conditions. (I–L) DAB staining, FRAP, CAT activity, and MDA content in leaves of ZH8015, oscata, and oscatc plants under control and PEG stress conditions. In (B–D,F–H,J–L), data are means ± SD (n = 3); ns, not significant; * p < 0.05, ** p < 0.01 determined by t-test; the test was performed on samples between the 9311 and OE/KO lines or between the ZH8015 and oscata/oscatc lines. Bars = 5 mm in (A,E,F).

Figure 6

Analysis of OsAP4 and OsCATA/OsCATC expression under hormone treatment. (A) Analysis of the promoter sequence of OsAP4. (B–D) Expression of OsAP4, OsCATA, and OsCATC in 9311 plants under PEG treatment conditions after being stimulated with IAA, GA, ABA, BL, JA, and CTK hormones. In (B–D), data are means ± SD (n = 3); ns, not significant; * p < 0.05, ** p < 0.01 determined by t-test; the test was performed on samples between the control and IAA/GA/ABA/BL/JA/CTK treatment.